Azathioprine (AZA) is a widely used immunosuppressive drug to avoid transplant rejection and control autoimmune disorders. In this study, rats were split into four groups, with one designated as the control. They were given ascending graded doses of AZA (6.25, 12.5, and 25 mg/kg body weight) orally, once daily for a month. Our results demonstrated that AZA caused a dose- dependent reduction in platelet count, prolonged bleeding time, clotting time, prothrombin, and activated partial thromboplastin times with high significant effects observed in the group receiving 25 mg/kg body weight. Hemato-toxicity was also illustrated, with the highest impact seen in the moderate and high dose groups. AZA progressively cause a reduction in serum total immunoglobulin (Ig) concentration, quantitative hemolysis of SRBCs, hemagglutination titer, delayed type hypersensitivity, splenocyte proliferation rate, and the percentage of CD3+CD8+ , CD3+CD4+ , and CD11b+ spleen cells in dose dependent way when compared to the control animals. Moreover, AZA ascending doses induced oxidative stress, renal and hepatic toxicity in a dose-dependent manner. This study is the first to highlight the examined ascending dose-dependent effects of AZA on rat hemostatic process, immune system, and organ functions. It emphasizes the importance of cautious monitoring and optimal dosing in clinical applications of AZA.