In the present study, the isolated compound Punicaflavone (PF) from the methanolic flower extract of Punica granatum (Linn.) was subjected to toxicity assessment in Wistar albino rats. Sub-acute toxicity studies were carried out by oral administration of the PF from Punica granatum (Linn.) for a period of 28 days at different dose levels of 50, 100 and 200mg/kg body weight. No major changes in body and organ weight, behavioural, haematology and histology were observed at the end of 28 days of daily oral administration. Biochemical parameters such as the levels of Glucose, Protein, Urea and Creatinine in serum were found to be well within the normal limits. Histopathological examination of the major vital organs (heart, liver and kidney) revealed no significant pathological alterations in the treated group of rats. The sub-acute toxicity studies of the PF showed no mortality and no symptoms of toxicity or behavioral changes at the maximum dose (1000mg/kg). The results indicate that the PF is safe up to a dose of 1000 mg/kg.