The present study was performed to assess the HPLC-DAD analysis as well as antioxidant and protective effects of Tunisian Rhanterium suaveolens (Rs) against acetamiprid (ACT) induced oxidative stress on mice erythrocytes. The in vitro assays showed that the methanolic extract of Rs has an impressive antioxidant effect proved by testing the total antioxidant and scavenging activities using BCB, DPPH and ABTS assays, respectively. Moreover, qualitative and quantitative analysis using HPLC-DAD revealed the richness of Rs in polyphenols where p-Coumaric, Apigenin-7-glucoside and Ferulic acid were detected as the most abundant polyphenols. In the in vivo experiment, ACT, used as a toxicity model, was given to mice at a dose of 20 mg/kg. The latter was the origin of hemolytic anemia characterized by a significant decrease in red blood cells, hemoglobin and hematocrit levels and an increase in bilirubin, LDH, osmotic fragility, reticulocytes and white blood cells number. Characteristic erythrocyte morphological alterations were also determined as spherocytosis, schistocytosis and dacryocystitis. The oxidative status of ACT-treated mice was also altered manifested by a significant increase in MDA and GSH levels and a decrease in SOD, CAT and GPx activities. When receiving the Rs methanolic extract at a dose of 300 mg/kg, all the parameters cited above were restored in mice. These remarkable corrections could only confirm the important antioxidant effect and the noticeable protective properties that possess Rs owing to its broad range of secondary bioactive metabolites.