Microplastics and nanoplastics (MP/NPs) in the environment were found not only affecting environmental health but also impacting human health. Humans are exposed to MP/NPs via several routes including oral ingestion, inhalation, and direct skin contact. MP/NPs can penetrate biological barriers and reach different parts of human body. In the current study, the potential toxicity and pathways induced by polystyrene nanoplastics (PS-NPs) on normal human cells were evaluated. The viability of human cells was determined upon exposure to distinct concentrations and sizes of PS-MP/NPs. The toxicity of PS-MP/NPs was correlated with the particle size, concentrations, and exposure duration. The toxic effects of 20 nm PS-NPs treatment on human embryonic lung fibroblast (MRC5), immortalised human keratinocytes (HaCaT), and nasopharyngeal epithelial (NP69) cells were further investigated. Flow cytometric analysis showed that MRC5 and HaCaT cells experienced S phase arrest while NP69 cells halted at G0/G1 phase in the treatment groups. Apoptosis was seen in MRC5 and NP69 cells post-PS-NPs exposure. JC-1 and DCFHDA fluorescence assays revealed depolarisation of mitochondrial membrane potential (MMP) and oxidative stress in all treatment groups. PS-NPs exposure caused mitochondrial dysfunction in the exposed cell lines. Reactome pathway analysis of the transcriptomic data revealed adipogenesis and carcinogenesis may be affected in the treatment groups of MRC5, HaCaT and NP69 cells. Gene set enrichment analysis (GSEA) suggested the activation of nuclear factor kappa B (NF-B) of NP69 cells upon exposure to PSNPs and increased mRNA expressions of NF-B targeted genes were validated. In conclusion, PS-NPs induced oxidative stress, mitochondrial disorder, cell cycle arrest, and apoptosis, in MRC5, HaCaT, and NP69 cells, possibly impact on lipid metabolism and lead to cancer progression. Transcriptomic study revealed that PS-NPs induced mitochondrial dysfunction and endoplasmic reticulum (ER) stress partly through the activation of NF-B pathway, which triggers inflammatory responses via tumour necrosis factor (TNF) and interferon (IFN) signalling in NP69 cells. Identification of pathway and mechanism which control cellular toxicity induced by PS-NPs may enable pinpointing therapeutic targets in managing adverse health effects induced by PS-NP.