Nanoplastics (NPs) are emerging environmental contaminants present in atmospheric, freshwater, and aquatic environments. NPs can rapidly permeate cell membranes and build up in human tissues and organs, causing a potential threat to human health. As the skeletal muscle undergoes aging, myogenesis gradually deteriorates, leading to loss of muscle mass. While previous studies have demonstrated the adverse and toxic effects of polystyrene (PS)-NPs, gaps remain in understanding aging effects and specific mechanisms by PS-NPs in pre-differentiated myoblasts. In this study, we investigated the cellular internalization, aggregation, and senescent effects of PS-NPs using an in vitro model of pre-differentiated C2C12 myoblasts. Pre-differentiated C2C12 myoblasts were exposed to increasing concentrations of PS-NPs and internalization was observed in myoblasts using flow cytometry and transmission electron microscopy (TEM). We further investigated whether internalization of these PS-NPs at sublethal cytotoxic concentrations led to an increase in senescence hallmarks, such as increased β-galactosidase activity, increased expression of p16, p21 and senescence-related secretory phenotypes, and cell cycle arrest. In addition, PS-NP treatment caused notable mitochondrial superoxide production and damage, including mitochondrial membrane depolarization, content loss, fragmentation, and decreased ATP production. Rotenone, a mitochondrial function inhibitor, and exacerbated PS-NP-induced cell proliferation inhibition, whereas Mito-TEMPO, a mitochondrial superoxide scavenger, restored the cell proliferation rate and rescued cellular senescence. Therefore, our findings indicate the senescent effects of PS-NPs through mitochondrial superoxide production and dysfunction in pre-differentiated myoblasts.