Human bladder contraction mainly depends on Ca²⁺ influx, via L-type voltage-gated Ca²⁺ channels, and on RhoA/Rho-kinase contractile signalling, which is up-regulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho-kinase signalling in rat and human bladder. Since in normal bladder from Sprague-Dawley (SD) rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness, and increased sensitivity to the L-type Ca²⁺ channel antagonist isradipine, we investigated whether elocalcitol up-regulated L-type Ca²⁺ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca²⁺], which was abrogated by the L-type Ca²⁺ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca²⁺ currents (ICa), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist Bay-K8644. Addition of elocalcitol (10^-7 M) increased L-type ICa size and specific conductance, by inducing faster activation and inactivation kinetics than control and Bay-K8644, while determining a significant negative shift of the activation and inactivation curves, comparable to Bay-K8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10^-8 M, 48h), which also showed increased mRNA and protein expression of pore forming L-Type 1C subunit. In bladder from SD rats, Bay-K8644 induced a dose-dependent increase in tension, that was significantly enhanced by elocalcitol treatment (30 µg/kg/day, 2 weeks). In conclusion, elocalcitol up-regulated Ca²⁺ entry through L-type Ca²⁺ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho-kinase signalling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.