SUMMARYMale obesity may lead to declines in testosterone levels, reproductive hormonal profile, and semen quantity. To assess the effects of obesity on spermatogenesis, Sprague–Dawley rats fed a high‐fat diet served as a model of induced obesity. The litter sizes for females mated to obese males were significantly lower as compared to females mated with normal‐diet‐fed controls. Their serum high‐density lipoprotein, low‐density lipoprotein, cholesterol, and estradiol levels increased in obese males, but testosterone and follicle‐stimulating hormone levels decreased. Testicular morphology disruptions included Sertoli‐cell atrophy, disrupted tight junctions, and mitochondrial degeneration in spermatogenic cells. To further investigate the molecular mechanisms leading to high‐fat‐diet‐induced changes, we employed testicular proteomic analysis on rats fed both types of diet. Three spots were up‐regulated in rats fed a high‐fat diet whereas two others were downregulated. One of the upregulated spots was palmitoyl‐protein thioesterase 1 (PPT1), a lipoprotein metabolizing related enzyme localized to Sertoli cells. In a Sertoli‐cell line cultured in a high‐fat supplemented medium, PPT1 abundance was accompanied by increases in the endocytic vesicle‐associated protein, clathrin, and decreases in the tight junctional proteins, ZO‐1 and occludin. In conclusion, declines in rat male fertility induced by a high‐fat diet are associated with an altered testicular protein expression pattern as well as disruption of testicular Sertoli‐cell and spermatogenic‐cell morphology. PPT1 expression may provide a testicular marker of reduced fertility in obese males, as increases in its expression may be detrimental to Sertoli‐cell function during spermatogenesis. Mol. Reprod. Dev. 81: 55–65, 2014. © 2013 Wiley Periodicals, Inc.