Microplastics have gained significant social attention, as they can enter our bodies through food and drinking water. The adrenal gland is essential for the maintenance of metabolic homeostasis and stress responses. Nevertheless, the effects of microplastics on the steroid synthesis in the adrenal cortex was still unclear. In this study, through both in vivo and in vitro models, we found that polystyrene microplastics (PS-MPs) impaired adrenal steroid synthesis, leading to a reduction in corticosterone levels. In vivo, we further observed that chronic exposure to PS-MPs (0.25, 0.5 and 1 mg/d for 4 weeks) could induce abnormal mitochondrial morphology and functional disruptions of adrenal glands in male mice, along with an imbalance in cellular oxidative stress, manifested as increased level of reactive oxygen species, diminished antioxidant activity (glutathione peroxidase and superoxide dismutase). In vitro, these occurrences coincided with an elevated rate of cell apoptosis observed in adrenocortical cells following exposure to PS-MPs. We proposed that mitochondrial dysfunction not only directly influenced the biosynthetic processes of steroid hormones but also induced cell apoptosis through the initiation of cellular oxidative stress. The latter may represent a common mechanism underlying the multi-organ toxicity induced by PS-MPs in the body. Our findings would provide new insights for the development of more effective environmental protection measures and the reduction of plastic pollution.