Perilla frutescens (L.) Britt. var. japonica (Hassk.) Hara (PF), is a medical herb of the Lamiaceae family. We have previously reported that the PF sprout extract (PFSE) is effective in treating hyperglycemia. However, the role of PFSE on glomerular mesangial cells (MCs) proliferation and the extracellular matrix (ECM) accumulation in a diabetic condition are still unclear. Therefore, in this study, we have investigated the role of PFSE on cell proliferation and ECM accumulation in murine glomerular MCs (MMCs), cultured under a high glucose (HG) condition. PFSE treatment attenuated HG-induced MMCs proliferation and hypertrophy. Moreover, the HG-induced ECM protein, collagen IV and fibronectin, overexpression was abolished by the PFFSE treatment. In addition, PFSE inhibited reactive oxygen species (ROS) overproduction and NOX2 and NOX4 expression in MMCs under a HG condition. Our data further revealed the involvement of mesangial cell damage in AMP-activated kinase (AMPK) activation. PFSE strongly activated AMPK in MMCs under hyperglycemic conditions. These results suggest that PFSE inhibits HG-medicated MC fibrosis through suppressing the activation of NOX2/4 and the AMPK activation mechanism. PFSE may be useful for the prevention or treatment of diabetic nephropathy.