Soy protein isolate (SPI) is a prevalent ingredient in various dairy, meat products, and health drinks. Concerns have been raised regarding potential adverse effects of soy consumption on male health, such as feminization and infertility. This concern is attributed to phytoestrogens, specifically isoflavones present in soy, acting as estrogen substitutes and modulators. This study aims to investigate the effects of soy protein isolate supplementation on testosterone hormone levels and the expression of genes associated with testosterone synthesis. Male rats (n = 18) were evenly divided into three groups: Group 1 (Normal Control) received oral administration of a saline vehicle. Group 2 (Low Dose) received 450 mg/kg body weight of soy protein isolate for 30 days. Group 3 (High Dose) received 900 mg/kg body weight of soy protein isolate for 30 days. All administrations were conducted intragastrically. Testosterone hormone levels were decreased with a disturbance in the mRNA expression of genes (Hsd17b6, SRD5A1, Akr1c3, Cyp17a1 and Hsd3b) involved in the biosynthesis of testosterone hormone in the low and high dose groups in in comparison to the control group. In the testes of the high dosage group, histopathological analysis showed a noticeable irregularity around the periphery of the seminiferous tubules. Our results have shown damage to testicular tissue and reduced genetic expression of certain genes responsible for the formation of testosterone hormones, followed by a reduction of testosterone hormones in the group of rats that were given a high dose of SPI for 30 days.