Tocotrienols (T3) are a family of vitamin E that exists as four isomers: α-, β-, γ-, and δ-T3. γ-T3 and δ-T3 reportedly displayed promising anticancer potential in two-dimensional (2D) cell culture model and immunomodulating effects in animal model of various cancer types, especially breast cancer. However, there is a lack of clinical trials evidence supporting the use of tocotrienols for breast cancer patients. The disparity between preclinical and clinical results posed as a huge challenge for the clinical application of tocotrienols. Hence, with the use of in vitro three-dimensional (3D) tumour spheroid model of breast cancer, this study aimed to gain deeper insight into the anticancer potential of γ-T3 and δ-T3 in the presence of a complex tumour microenvironment. Tumour spheroid serves as a mimetic preclinical model due to their similarity with breast tumour characteristics. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic options. Two cell lines of different TNBC subtypes were employed: (1) MDA-MB-231, a mesenchymal-like subtype and (2) HCC38, a basal-like 1 subtype of TNBC. Tumour spheroid were successfully generated using Nunclon Sphera 96-Well U-Shaped-Bottom microplate and validated by morphology observation and gene expressions of 3D spheroids biomarkers using RT-qPCR, which were rigorously selected using data mining based on published studies of breast tumour spheroid. Notably, CD44, MMP1 and VEGFA were significantly upregulated in TS-MDA-MB-231 compared to 2D monolayer cells. Similarly, MMP1 and VEGFA were significantly upregulated in TS-HCC38. Cell proliferating assays (WST-1, CCK-8, and PrestoBlue HS) demonstrated that δ-T3 and γ-T3 were cytotoxic to the tumour spheroids and monolayer model derived from TNBC cells. The half-maximal growth inhibitory dose (GI50) of δ-T3 and γ-T3 in each model were determined. δ-T3 and γ-T3 were able to significantly and dose-dependently inhibit the viability of tumour spheroid in TS-MDA-MB-231 (δ-T3 27.44 μg/mL; γ-T3 19.44 μg/mL) and TS-HCC38 (δ-T3 38.21 μg/mL; γ-T3 19.44 μg/mL) , model albeit at a higher dosage compared to the monolayer model of MDA-MB-231 (δ-T3 11.53 μg/mL; γ-T3 12.01 μg/mL) and HCC38 (δ-T3 5.68 μg/mL; γ-T3 6.35 μg/mL). δ-T3 and γ-T3 induced ultrastructural changes in the tumour spheroid, including an increase in area, diameter and volume, as well as a decrease in circularity and solidity indicating cellular disaggregation. Total RNA of tocotrienols (δ-T3 or γ-T3) treated and untreated TS-MDA-MB-231 and TS-HCC38 were extracted with the Zymo Quick-RNA Miniprep Kit for transcriptome-level expression profiling using the Clariom D Affymetrix GeneChip platform. Tocotrienols (δ-T3 or γ-T3) regulated a total of 2567 and 5476 differentially expressed genes (DEGs) in δ-T3 and γ-T3 treated TS-MDA-MB-231, respectively. In TS-HCC38, 1004 and 278 DEGs were identified in response to δ-T3 and γ-T3 treatment, respectively, compared to untreated controls. In-depth functional bioinformatics analysis (Gene Ontology and KEGG pathway) of the transcriptomic profile revealed TNBC subtype-specific effects of tocotrienols in several pathways. Additionally, tocotrienols regulated a subset of long non-coding RNAs in TS-MDA-MB-231 which were further verified via comparison with the expression level of target mRNA. Notably, tocotrienol isoforms (δ-T3 or γ-T3) induced differential expression in pathways (oxidative phosphorylation, cell cycle, ribosome, proteasome), which were significantly downregulated in TS-HCC38, but an opposite upregulated effect of the same pathways was observed in TS-MDA-MB-231. Disease association analysis of the transcriptome profile revealed tocotrienol modulated clinically relevant pathways in breast cancer, δ-T3 significantly downregulated genes associated with cell cycle pathway, whereas γ-T3 downregulated genes associated with steroid hormone biosynthesis pathway in TS-HCC38. However, genes associated with cell cycle control and DNA damage response were upregulated in TS-MDA-MB-231. RT-qPCR validation demonstrated that both δ-T3 and γ-T3 differentially regulate MMP1, a key angiogenesis regulator and tumour spheroid biomarker, depending on the TNBC cell line used. Findings from this study suggest that tailoring tocotrienol treatment based on specific TNBC subtype is warranted to optimise therapeutic outcomes.