Increased mesangial cell proliferation is one of the major pathologic features in the early stage of diabetic nephropathy (DN). Carnosine is an endogenously synthesized dipeptide that has been reported as a protective factor in diabetic nephropathy. However, the underlying mechanism involved in this effect remains to be elucidated. In this study, the effect of carnosine on cell proliferation and its underlying mechanisms were investigated in cultured rat mesangial cells by the methylthiazoletetrazolium (MTT) assay, the 5-bromo-2-deoxy-uridine (BrdU) cell proliferation assay, flow cytometry and western blotting. The results showed that pretreatment of mesangial cells with carnosine significantly inhibited cell proliferation and DNA synthesis in a dose-dependent manner by increasing the cell population in G1 and reducing that in S-phase. In addition, carnosine could reverse high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21 but not that of p27. Furthermore, carnosine could reduce the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK). Taken together, these results suggest that carnosine can inhibit mesangial cell proliferation by modulating cell cycle progress, indicating that carnosine could be a potential therapeutic agent for the prevention of DN in the early stage.