Root knot nematodes (RKNs, Meloidogyne spp.) are microscopic roundworms with a wide host range causing great economic losses worldwide. Understanding how metabolic pathways function within the plant upon RKN infection will provide insight into the molecular aspects of plant-RKN interactions. Glucose-6-phosphate dehydrogenase (G6PDH), the key regulatory enzyme of the oxidative pentose phosphate pathway (OPPP), is involved in plant responses to abiotic stresses and pathogenesis. In this study, the roles of Arabidopsis cytosolic G6PDH in plant-RKN interactions were investigated. Enzyme assays and western blotting were used to characterize changes in total G6PDH activity and protein abundance in wild-type Arabidopsis in response to RKN infection. The susceptibility of wild-type plants and the double mutant g6pd5/6 to RKNs was analysed and the expression of genes associated with the basal defence response was tested after RKN infection using quantitative reverse transcription PCR. RKN infection caused a marked increase in total G6PDH activity and protein abundance in wild-type Arabidopsis roots. However, the transcript levels of G6PDH genes except G6PD6 were not significantly induced following RKN infection, suggesting that the increase in G6PDH activity may occur at the post-transcriptional level. The double mutant g6pd5/6 with loss-of-function of the two cytosolic isoforms G6PD5 and G6PD6 displayed enhanced susceptibility to RKNs. Moreover, reactive oxygen species (ROS) production and gene expression involved in the defence response including jasmonic acid and salicylic acid pathways were suppressed in the g6pd5/6 mutant at the early stage of RKN infection when compared to the wild-type plants. The results demonstrated that the G6PDH-mediated OPPP plays an important role in the plant-RKN interaction. In addition, a new aspect of G6PDH activity involving NADPH production by the OPPP in plant basal defence against RKNs is defined, which may be involved in ROS signalling.