Transient receptor potential cation channel 6 (TRPC6) is one of the key molecules for filtration barrier function of podocytes. Over-expression of TRPC6 in podocytes is frequently found in acquired or inherited proteinuric kidney diseases, and animal model over-expression of TRPC6 may lead to proteinuria. To investigate the impact of TRPC6 over-expression in podocytes on its function and its relation to proteinuria in kidney diseases, we over-expressed TRPC6 in mouse podocytes by transient transfection of TRPC6 cDNA plasmid, and observed their changes in foot processes, intracellular F-actin distribution, nephrin and synaptopodin expression, electrophysiology, RhoA activity and intracellular Ca(2+). In podocytes over-expressing TRPC6, cell processes were reduced remarkably in association with the derangement of cytoskeleton demonstrated by the abnormal distribution of intracellular F-actin. These cells also displayed a higher increase of intracellular Ca(2+) ion to the TRPC6 agonist 1-oleoyl-acetyl-sn-glycerol and a higher current in the patch-clamp experiment, down-regulation of nephrin and synaptopodin expression and increase of activated RhoA. These changes could be rescued by the treatment of the cells with U73122 to block TRPC6 channel or BAPTA-AM to chelate intracellular Ca(2+) ion. Additionally, the podocytes over-expressing TRPC6 treated with RhoA inhibitor Y-27632 showed an improvement in F-actin arrangement in the cells and increase of nephrin and synaptopodin expression. From these results, we therefore propose that over-expression of TRPC6 in podocytes may be one of the fundamental changes relating to the dysfunction of the slit diaphragm and proteinuria. Podocytes over-expressing TRPC6 may lead to higher intracellular Ca(2+) ion concentration in the presence of stimuli. The increase of intracellular Ca(2+) down-regulates the expression of two important molecules, nephrin on slit diaphragm and synaptopodin in cytoskeleton, and stimulates RhoA activity, which in turn causes F-actin derangement and the decrease of foot processes.