Background: Glucose-6-phosphate dehydrogenase (G6PD) participates in glucose utilization by catalysing the first step of the pentose-phosphate pathway in mammalian cells. Previous studies have shown that changes in G6PD levels can promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in a human melanoma xenograft model. G6PD cooperates with NADPH oxidase 4 (NOX4) in the cellular metabolism of reactive oxygen species (ROS) and in maintaining the intracellular redox state. Methods: In this study, the effect of G6PD or NOX4 silencing in the melanoma line A375 was examined in terms of redox state, proto-oncogene tyrosine-protein kinase Src (c-Src) and the tyrosine-specific protein phosphatase SHP2 expression as well as cell cycle progression. Results: The results demonstrate that: (1) Downregulation of cyclin D1 and CDK4 and up-regulation of p53 and p21 occurred in response to silencing of G6PD and NOX4 thus resulting in G1/S cell cycle arrest and inhibition of A375 cell proliferation. (2) The blockade of cell proliferation is primarily due to a reduced DNA-binding activity of STAT3. (3) The DNA-binding activity of STAT3 was regulated by the upstream factors, c-SRC and SHP2. Silencing of NOX4 in A375 cells inhibited c-SRC and SHP2 regulated STAT3 activity. Conclusion: The data are consistent with a novel G6PD-NOX4-NADPH-ROS-c-SRC/SHP2 pathway controlling STAT3 activity in A375 melanoma cells.