Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme in pentose phosphate pathway (PPP), excessive activation of which has been considered to be involved in tumorigenesis. Here, we show that tyrosine kinase c-Src interacts with and phosphorylates G6PD at Tyr 112. This phosphorylation enhances catalytic activity of G6PD by dramatically decreasing its Km value and increasing its Kcat value for substrate glucose-6-phosphate. Activated G6PD therefore augments the PPP flux for NADPH and ribose-5-phosphate production which is required for detoxification of intracellular reactive oxygen species (ROS) and biosynthesis of cancer cells, and eventually contributes to tumorigenesis. Consistently, c-Src activation is closely correlated with tyrosine phosphorylation and activity of G6PD in clinical colorectal cancer samples. We thus uncover another aspect of c-Src in promoting cell proliferation and tumorigenesis, deepening our understanding of c-Src as a proto-oncogene.