Obesity and overweight are frequently associated with male subfertility. To address new findings on the players involved in the obesity-induced impairment of spermatogenesis, we used a high-fat diet-induced overweight-rat model. Following four weeks of high-fat diet, the organization of seminiferous epithelium was affected, and tubules lumen showed immature/degenerated cells, typical signs of hormonal imbalance and testicular damage. Real-time PCR analysis allowed us to detect increased levels of ERα and decreased levels of aromatase CYP19 transcripts in testis, suggesting an increase in circulating estrogens derived from the accumulating adipose tissue rather than the induction of testicular estrogen synthesis. Moreover, in situ hybridization analysis showed an increased susceptibility towards estrogens in testis from high-fat fed rats, being ERs expressed not only in spermatogonia, as in control testis, but also in spermatids. Western blot and immunohistochemical analyses revealed an increase in the amount of p53 and PCNA, together with a change in their immunodetection, being the proteins localised on germ cells at different stages of maturation. Differences in p53 and PCNA expression may give evidence and be part of a cellular response to stress conditions and damage caused by the excessive intake of saturated fatty acids.