Sexual differentiation in the brain takes place from late gestation to the early postnatal days. This is dependent on the conversion of circulating testosterone into estradiol by the enzyme aromatase. The glyphosate was shown to alter aromatase activity and decrease serum testosterone concentrations. Thus, the aim of this study was to investigate the effect of gestational maternal glyphosate exposure (50 mg/kg, NOAEL for reproductive toxicity) on the reproductive development of male offspring. Sixty-day-old male rat offspring were evaluated for sexual behavior and partner preference; serum testosterone concentrations, estradiol, FSH and LH; the mRNA and protein content of LH and FSH; sperm production and the morphology of the seminiferous epithelium; and the weight of the testes, epididymis and seminal vesicles. The growth, the weight and age at puberty of the animals were also recorded to evaluate the effect of the treatment. The most important findings were increases in sexual partner preference scores and the latency time to the first mount; testosterone and estradiol serum concentrations; the mRNA expression and protein content in the pituitary gland and the serum concentration of LH; sperm production and reserves; and the height of the germinal epithelium of seminiferous tubules. We also observed an early onset of puberty but no effect on the body growth in these animals. These results suggest that maternal exposure to glyphosate disturbed the masculinization process and promoted behavioral changes and histological and endocrine problems in reproductive parameters. These changes associated with the hypersecretion of androgens increased gonadal activity and sperm production.