Background: Enicostemma axillare (Lam.) Raynal is used in traditional practice for the treatment of diabetes, malaria and liver disorders. No systematic toxicity study was described for this plant and hence the present was undertaken to evaluate acute and sub-acute toxicity of aqueous extract of Enicostemma axillare (AEEA). Objective: The acute oral toxicity study of AEEA was carried out as per the OECD guidelines 423 in mice and the sub-acute toxicity was carried out as per the guidelines set by OECD 407 in male and female rats. Materials and Methods: Body weight, food and water consumption, hematological parameters, biochemical parameters, organ weight and histopathological analysis were carried out. Results: No gross toxicity and mortality was observed upto a dose of 2000 mg/kg. For sub-acute toxicity test, 200 mg/kg and 400 mg/kg daily dose of AEEA administered orally for 28 days in male and female group of rats not exhibited any signs of toxicity and mortality.