Canonical transient receptor potential-6 channels (TRPC6) have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during phospholipase C (PLC)-dependent transduction cascades. TRPC6 can also be activated by reactive oxygen species (ROS). We have previously shown that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally co-immunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91phox). The NOX2-TRPC6 interaction was not detected in cells stably expressing an shRNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane permeable DAG analog (OAG) increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47phox. In whole-cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium (DPI) and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts using methyl-β-cyclodextrin (MBCD) reduced activation of podocyte TRPC6 channels by OAG, and also eliminated the NOX2-TRPC6 interaction as assessed by co-immunoprecipitation. These data suggest that active NOX2 complex assembles with TRPC6 at podocin-organized sterol-rich raft domains, and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.