Connexin 43 (Cx43) plays an important role in cell differentiation and growth control, but whether it can be regulated by high glucose and whether it can mediate in glomerular mesangial cells (GMC) the phenotype alterations that are induced by high glucose still remain to be explored. In this study, RNA interference and gene transfer techniques were used to knock down and overexpress Cx43 gene in rat GMC to determine the contribution of Cx43 to GMC senescence that was induced by high glucose. The results show that high glucose (30 mM) not only downregulated Cx43 mRNA and protein expression (P<0.05) but also increased the percentage of senescence-associated beta-galactosidase (SA-beta-gal) stained cells and expression of p21cip1 and p27kip1 (P<0.05), indicating that high glucose promoted rat GMC senescence. Knocking down Cx43 gene expression significantly increased the percentage of SA-beta-gal stained cells and p27kip1 and p21cip1 expression in GMC (P<0.05), whereas overexpression of Cx43 significantly decreased the percentage of SA-beta-gal stained cells (P<0.05). These results demonstrate for the first time that downregulation of Cx43 expression by high glucose promotes the senescence of GMC, which may be involved in the pathogenesis of diabetic nephropathy.