Microplastics enter the human body through plastic food packaging, food products, drinking water, and contaminated air. Microplastics produce negative impact on fertility of both males and females. In males, microplastics have been found to reduce sperm quality, testicular function, and overall fertility. The present study investigated the effects of polystyrene microplastics (PS-MPs) on the epididymis and seminal vesicle. Male Wistar albino rats were divided into groups based on administered doses of PS-MPs: 5, 50, 500, and 5000 μg per kilogram of body weight. A sham control group was included for comparison. The study assessed organ weight, sperm characteristics, histological and biochemical parameters. The results revealed a dose-dependent decline in epididymal weight, ranging from 3% to 11%. Sperm analysis showed a significant decline in sperm count, viability, and motility across all test groups. Morphological assessments of cauda epididymal sperm revealed higher prevalence of defects, including, bent tails, coiled tails, headless tails, and tail-less heads. Histological examination highlighted pronounced structural aberrations in the epididymis and seminal vesicle, particularly in animals treated with the highest dose of PS-MPs. Biochemical analysis showed a 35% reduction in sialic acid levels and diminished L-carnitine activity, indicating functional impairment of the epididymis. Additionally, reduced fructose levels in the seminal vesicle suggested a decline in the energy available in seminal fluid for ejaculated sperm. Overall, the study concluded that PS-MPs have a significant negative impact on the epididymis and seminal vesicle, likely affecting sperm maturation and post-ejaculation efficiency.