We investigated the effects of sub-chronic exposure to Kalach 360 SL (KL), glyphosate-based herbicide used in Tunisia, on liver and hematological system in different groups of female rats. Group 1 was used as a control, while animals of groups 2 and 3 received orally 0.07 mL and 0.175 mL of KL, respectively (126 and 315 mg of glyphosate/kg), for 60 days. As a result, the KL-exposed groups exhibited hypochromic microcytic anemia, systemic inflammation, cytolysis, decrease in hepatic enzyme activity, and cholestasis. Exposure to different doses of KL could induce erythrocyte destruction (hemolysis) in hematopoietic organs (bones). Moreover, lipid peroxidation contents and protein oxidation markers significantly increased in exposed groups, while enzymatic and non-enzymatic antioxidant activities decreased considerably, in both erythrocytes and liver tissues, compared with those in controls. Liver histological studies confirmed the presence of inflammatory reaction with pathology involving the damage or necrosis of hepatocytes, however, without fibrosis remodulation. Thus, KL sub-chronic exposure caused hepatonecrosis, systemic inflammation, and hemolysis.