Antiretroviral therapy has effectively suppressed HIV infection and replication and prolonged the lifespan of HIV-infected individuals. In the meantime, various complications including type 2 diabetes associated with long-term antiviral therapy have shown steady increases. Metformin has been the front-line anti-hyperglycemic drug of choice and the most widely prescribed medication for the treatment of type 2 diabetes. However, little is known about the effects of Metformin on HIV infection and replication. In this study, we showed that Metformin treatment enhanced HIV gene expression and transcription in HIV-transfected 293T and HIV-infected Jurkat and human PBMC. Moreover, we demonstrated that Metformin treatment resulted in increased CREB expression and phosphorylation, and TBP expression. Furthermore, we showed that Metformin treatment increased the recruitment of phosphorylated CREB and TBP to the HIV LTR promoter. Lastly, we showed that inhibition of CREB activation significantly abrogated Metformin-enhanced HIV gene expression. Taken together, these results demonstrated that Metformin treatment increased HIV transcription, gene expression, and production though increased CREB phosphorylation and recruitment to the HIV LTR promoter. These findings may help design the clinical management plan and HIV cure strategy of using metformin to treat type 2 diabetes, a comorbidity with an increasing prevalence, in people living with HIV.