The current study aimed to investigate the effects of thymoquinone (TQ) to enhance the anti-diabetic effect of metformin (MET) in streptozotocin-induced diabetes in male rats. The mutual effect of TQ and MET on biochemical parameters such as glucose, cholesterol, triglycerides, and glycated hemoglobin was estimated. Also, their combined influence on malonaldehyde (MDA) and total anti-oxidant capacity (TAC) in liver and pancreas tissues homogenates were assessed. Furthermore, their synergistic effects on the expression of mRNA encoding glucose transporter-2 (Glut-2) were evaluated. Moreover, insulin receptor labeling index percentage in liver tissues was estimated. Results revealed that the alteration in biochemical levels due to streptozotocin action was corrected and restored by TQ plus MET with more pronounced effect than MET alone. Also, in liver homogenate both TQ and MET decreased the elevated MDA level to 8.82 and augmented the TAC level to 2.71, compared to their values in MET group 14.64 and 1.19, respectively. Furthermore, TQ plus MET up-regulated the expression level of Glut-2 by 88.2%, compared to 80% in MET group. Taken together, our study revealed that TQ via its anti-oxidant properties is a useful combination therapeutic agent to enhance the anti-diabetic activity of MET in STZ-induced diabetic rats.