Glucose-6-phosphate dehydrogenase (G6PD) is crucial rate-limiting enzyme of the pentose phosphate pathway (PPP). G6PD dysregulation has been reported in various types of human cancer, and the role of G6PD in cancer progression was demonstrated in numerous studies. A previous study from our laboratory described the prognostic significance of G6PD in clear cell renal cell carcinoma (ccRCC), and demonstrated its proliferative role through positive feedback regulation of the phosphorylated form of signal transducer and activator of transcription 3. However, the role of G6PD in ccRCC invasion remains unclear. In the present study, reverse transcription-quantitative (RT-q) PCR, western blotting, enzyme activity assay, transwell assay and immunohistochemistry analysis in cell model, xenograft mice model and human specimen studies were performed to evaluate the role of G6PD in ccRCC invasion. The results from the present study demonstrated that G6PD may promote ccRCC cell invasive ability by increasing matrix metalloproteinase 2 (MMP2) mRNA and protein expression both in vitro and in vivo. In addition, a positive correlation between G6PD and MMP2 expression was demonstrated by RT-qPCR and western blotting in twenty pairs of ccRCC tumor specimens and matched adjacent normal tissues. Furthermore, G6PD promoted reactive oxygen species (ROS) generation and activated the MAPK signaling pathway in ccRCC cells. In addition, ROS significantly promoted the MAPK signaling pathway activation, which in turn contributed to MMP2 overexpression in ccRCC cells. In conclusion, the present study demonstrated that G6PD may facilitate ccRCC cell invasive ability by enhancing MMP2 expression through ROS-MAPK axis pathway. © 2020 Spandidos Publications. All rights reserved.