In high glucose, glomerular mesangial cells (MCs) demonstrate impaired Ca2+ signaling in response to seven-transmembrane receptor stimulation. To identify the mechanism, we first postulated decreased release from intracellular stores. Intracellular Ca2+ was measured in fluo-3-loaded primary cultured rat MCs using confocal fluorescence microscopy. In high glucose (HG) 30 mM for 48 h, the 25 nM ionomycin-stimulated intracellular Ca2+ response was reduced to 82% of that observed in normal glucose (NG). In NG 5.6 mM, Ca2+ responses to endothelin (ET)-1 and platelet-derived growth factor (PDGF) were unchanged in cells cultured in 50 nM Ca2+ vs. 1.8 mM Ca2+. Depletion of intracellular Ca2+ stores with thapsigargin eliminated ET-1-stimulated Ca2+ responses. Incubation in 30 mM glucose (HG) for 48 h or stimulation with phorbol myristate acetate (PMA) for 10 min eliminated the Ca2+ response to ET-1 but had no effect on the PDGF response. Downregulation of protein kinase C (PKC) with 24-h PMA or inhibition with Gö6976 in HG normalized the Ca2+ response to ET-1. Because ET-1 and PDGF stimulate Ca2+ signaling through different phospholipase C pathways, we hypothesized that, in HG, PKC selectively phosphorylates and inhibits PLC-β3. Using confocal immunofluorescence imaging, in NG, a 1.6- to 1.7-fold increase in PLC-β3 Ser1105 phosphorylation was observed following PMA or ET-1 stimulation for 10 min. In HG, immunofluorescent imaging and immunoblotting showed increased PLC-β3 phosphorylation, without change in total PLC-β3, which was reversed with 24-h PMA or Gö6976. We conclude that reduced Ca2+ signaling in HG cannot be explained by reduced Ca2+ stores but is due to conventional PKC-dependent phosphorylation and inactivation of PLC-β3.