Glomerular mesangial cell (MC) hypertrophy is regarded as one of the earliest pathological characteristics of diabetic nephropathy (DN), which plays a critical role in the pathogenesis of glomerulosclerosis. This study investigated the role of microRNAs (miRNAs) in MC hypertrophy due to exposure to high glucose. With a microarray, we screened the differential profiles of miRNAs in the renal cortex of DN mice, as verified by reverse transcription PCR with subsequent analysis of bioinformatics. We found miR-196a was downregulated remarkably in DN mice and increased the hypertrophy-related gene of p27(kip1) in high-enrichment gene ontologies. Furthermore, transfection of the miR-196a mimic greatly inhibited the expression of p27(kip1) with recovery of MC hypertrophic morphology. With flow cytometry, we also found that overexpression of miR-196a significantly reduced the percentage of G(1) phase arrest in the cell cycle. Cotransfection of the miR-196a mimic with a wild type of 3 UTR of the p27(kip1) vector reduced the activity of the luciferase reporter significantly in contrast to the miR-196a mimic with a mutant of the counterpart in HEK293 cell lines, suggesting that miR-196a directly targets p27(kip1). Finally, knockdown of p27(kip1) with specific small interfering RNA in MCs substantially reversed MC hypertrophy induced by transfection of the miR-196a inhibitor. This study revealed that miR-196a acts as an important molecular regulator in high glucose-induced MC hypertrophy by targeting p27(kip1).