The present study was conducted to determine whether and how store-operated Ca²⁺ entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7 day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca²⁺ channel. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca²⁺ currents were significantly enhanced in the MCs treated with HG for 7 days and the enhanced response was abolished by both GSK-7975A and La3+. In contrast, receptor-operated Ca²⁺ entry in MCs was significantly reduced by HG treatment. Western blot showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real time RT-PCR revealed that a 6 day HG treatment significantly increased mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription, but increases Orai1 protein through a post-transcriptional mechanism.