Alteration of [Ca<sup>2+</sup>]<sub>i</sub> by hyperglycemia is implicated in the pathogenesis of diabetic nephropathy. However, the effect of high glucose on Ca<sup>2+</sup> regulation in proximal tubule cells is not known. Thus, we examined the mechanisms by which high glucose regulates Ca<sup>2+</sup> uptake in primary cultured rabbit renal proximal tubule cells. Glucose increased the Ca<sup>2+</sup> uptake in a time– and dose–dependent manner. A stimulatory effect of high glucose on Ca<sup>2+</sup> uptake is predominantly observed using 25 m M glucose (high glucose) after 1 h, while 25 m M glucose did not affect cell viability and lactate dehydrogenase release. However, 25 m M mannitol and L–glucose did not affect Ca<sup>2+</sup> uptake as compared with controls. Nifedipine and methoxyverapamil (L–type Ca<sup>2+</sup> channel blockers) blocked high–glucose–induced stimulation of Ca<sup>2+</sup> uptake. High–glucose–induced stimulation of Ca<sup>2+</sup> uptake was blocked by pertussis toxin, SQ–22536 (adenylate cyclase inhibitor), myristoylated amide 14–22 (protein kinase A inhibitor), neomycin and U–73122 (phospholipase C inhibitors), and staurosporine and bisindolylmaleimide I (protein kinase C inhibitors). In addition, KN–62 (a Ca<sup>2+</sup>/calmodulin–dependent protein kinase II inhibitor) and W–7 (a Ca<sup>2+</sup>/calmodulin antagonist) blocked high–glucose–induced stimulation of Ca<sup>2+</sup> uptake. In conclusion, high glucose stimulates the Ca<sup>2+</sup> uptake through L–type Ca<sup>2+</sup> channels via G–protein–coupled adenylate cyclase/cAMP and phospholipase C/protein kinase C pathways.