Atrazine is a herbicide commonly used on grain crops. It has been identified as a potential endocrine disruptor, especially in freshwater species. However, there is little research on its endocrine disruptor effect in crustaceans. The current study was designed to estimate the median lethal concentration of atrazine (96-h LC50) and to assess the response of the freshwater crayfish Procambarus clarkii as a bioindicator for endocrine disruptors by measuring levels of steroid hormones, total protein, changes in Cytochrome P450 1B1, and histopathology of the ovary and testis. The results showed that the 96-h LC50 of atrazine for male and female P. clarkii was 10.62 and 12.66 mg L −1, respectively. LC10 (1.06 and 1.27mg/l) and LC25 (2.66 and 3.17 mg/l) values for males and females, were chosen for sublethal studies for 28 days. The results indicated that females are more tolerant to atrazine than males. Testosterone and total protein levels were significantly lower, while estradiol and progesterone levels were significantly higher in atrazine-exposed crayfish compared to the control. Also, the levels of CYP450 1B1 increased significantly in the testis, ovary, and hemolymph. The most noticeable histopathological changes in the testis were distorted architecture, spermatogonia distribution disruption in some testicular acini, Vacuolation, and hyperplasia. In the ovary, the separation between the ovarian epithelium and oogenetic pouch and lysis in the ovarian epithelium was noted. These parameters could be used as biomarkers to assess herbicide toxicity. Furthermore, P. clarkii could be used as an indicator of endocrine-disrupting chemicals in waterways.