Increased FGF23 levels are an early pathological feature in chronic kidney disease (CKD), causing increased cardiovascular risk. The regulation of FGF23 expression is complex and not completely understood. Thus, Ca²⁺ has been shown to induce an increase in FGF23 expression, but whether that increase is mediated by simultaneous changes in parathyroid hormone (PTH) and/or vitamin D is not fully known. Osteoblast-like cells (OLCs) from vitamin D receptor (VDR)^+/+ and VDR^−/− mice were incubated with Ca²⁺ for 18 h. Experimental hypercalcemia was induced by calcium gluconate injection in thyro-parathyroidectomized (T-PTX) VDR ^+/+ and VDR^−/− mice with constant PTH infusion. Inorganic Ca²⁺ induced an increase in FGF23 gene and protein expression in osteoblast-like cells (OLCs), but the increase was blunted in cells lacking VDR. In T-PTX VDR ^+/+ and VDR^−/− mice with constant PTH levels, hypercalcemia induced an increase in FGF23 levels, but to a lower extent in animals lacking VDR. Similar results were observed in FGF23 expression in bone. Renal and bone 1α-hydroxylase expression was also modulated. Our study demonstrates that Ca²⁺ can increase FGF23 levels independently of vitamin D and PTH, but part of the physiological increase in FGF23 induced by Ca²⁺ is mediated by vitamin D signaling.