Nanoplastics can be ingested by organisms and penetrate biological barriers to affect multiple physiological functions. However, few studies have focused on the effects of nanoplastics on the mammalian immune system. We evaluated the effects and underlying mechanism of nanoplastics of varying particle sizes and surface charges on murine splenic lymphocytes. We found that nanoplastics penetrated into splenic lymphocytes and that nanoplastics of a diameter of 50 nm were absorbed more efficiently by the cells. The nanoplastics decreased cell viability, induce cell apoptosis, up-regulated apoptosis-related protein expression, elicited the production of reactive oxygen species, altered mitochondrial membrane potential, and impaired mitochondrial function. Positively charged nanoplastics exerted the strongest toxicity. Negatively charged and uncharged nanoplastics caused oxidative stress and mitochondrial structural damage in lymphocytes, while positively charged nanoplastics induced endogenous apoptosis directly. Moreover, nanoplastics inhibited the expression of activated T cell markers on the T cell surface, while inhibiting the differentiation of CD8⁺ T cells and the expression of helper T cell cytokines. In terms of the mechanism, a series of key signaling molecules in the pathways of T cell activation and function were markedly down-regulated after exposure to nanoplastics.