In clinical practice, as there arises several situations, where a diabetic patient receiving pioglitazone need to be administered with an antifungal drug like Ketoconazole. However, the influence of Ketoconazole on antidiabetic activity of above mentioned drugs has not yet been properly studied and reported. Hence, keeping the possibility of interactions between Ketoconazole and oral anti-diabetic agents in view, the present study was planned to study the influence of Ketoconazole pretreatment on the anti-diabetic activity of oral antidiabetic agents. Materials and methods: The whole study was divided into 3 phases. 1. Influence of ketoconazole on blood glucose levels in normal rat and rabbits. 2. Influence of ketoconazole pretreatment (1week) on the hypoglycemic activity of oral anti-diabetic agent in normal rat and rabbits. 3. Influence of ketoconazole pretreatment (1week) on the anti-diabetic activity of oral anti-diabetic agent in streptozotocin induced diabetic rats. The blood glucose level was estimated with the help of GOD/POD method. Results and Discussion: Ketoconazole pre-treatment for one week has significantly preponed the onset of hypoglycemia and enhanced the peak hypoglycemic effect of pioglitazone in normal rats/rabbits and diabetic rats. The potentiation of hypoglycaemic effects of pioglitazone by the ketoconazole pretreatment is due to the inhibition of CYP3A4 and CYP2C8 isoenzymes by ketoconazole.