Elevated palmitic acid, a most abundant saturated fatty acid, is a risk factor for obesity complications. Here, we report that palmitic acid impairs spermatogenesis by inducing abnormal palmitoylation in Sertoli cells. Firstly, we proposed a correlation between palmitic acid and spermatogenesis by reporting a significant elevation of serum palmitic acid levels in patients with dyszoospermia. In palmitic acid -treated mice, the blood-testis barrier was found to be disrupted, and decreased tight junction protein expression was observed in palmitic acid-treated Sertoli cells. Palmitic acid entered the endoplasmic reticulum and induced endoplasmic reticulum stress, resulting in damage to Sertoli cell barriers.