Aflatoxicosis is a grave threat to the poultry industry. Dietary supplementation with antioxidants showed a great potential in enhancing the immune system; hence, protecting animals against aflatoxin B1-induced toxicity. Grape seed proanthocyanidin extract (GSPE) one of the most well-known and powerful antioxidants. Therefore, the purpose of this research was to investigate the effectiveness of GSPE in the detoxification of AFB1 in broilers. A total of 300 one-day-old Cobb chicks were randomly allocated into five treatments of six replicates (10 birds per replicate), fed ad libitum for four weeks with the following dietary treatments: 1. Basal diet (control); 2. Basal diet + 1 mg/kg AFB1 contaminated corn (AFB1); 3. Basal diet + GSPE 250 mg/kg; (GSPE 250 mg/kg) 4. Basal diet + AFB1 (1 mg/kg) + GSPE 250 mg/kg; (AFB1 + GSPE 250 mg/kg) 5. Basal diet + AFB1 (1mg/kg) + GSPE 500 mg/kg, (AFB1 + GSPE 500 mg/kg). When compared with the control group, feeding broilers with AFB1 alone significantly reduced growth performance, serum immunoglobulin contents, negatively altered serum biochemical contents, and enzyme activities, and induced histopathological lesion in the liver. In addition, AFB1 significantly increased malondialdehyde content and decreased total superoxide dismutase, catalase, glutathione peroxide, glutathione-S transferase, glutathione reductase activities, and glutathione concentration within the liver and serum. The supplementation of GSPE (250 and 500 mg/kg) to AFB1 contaminated diet reduced AFB1 residue in the liver and significantly mitigated AFB1 negative effects. From these results, it can be concluded that dietary supplementation of GSPE has protective effects against aflatoxicosis caused by AFB1 in broiler chickens.