Opening of large conductance calcium-activated and voltage-dependent potassium (BK) channels hyperpolarizes plasma membranes of smooth muscle (SM) to cause vasodilation, underling a key mechanism for mediating uterine artery (UA) dilation in pregnancy. Hydrogen sulfide (HS) has been recently identified as a new UA vasodilator, yet the mechanism underlying HS-induced UA dilation is unknown. Here, we tested whether HS activated BK channels in human UA smooth muscle cells (hUASMC) to mediate UA relaxation. Multiple BK subunits were found in human UA in vitro and hUASMC in vitro, and high β1 and γ1 proteins were localized in SM cells in human UA. Baseline outward currents, recorded by whole-cell and single-channel patch clamps, were significantly inhibited by specific BK blockers iberiotoxin (IBTX) or tetraethylammonium, showing specific BK activity in hUASMC. HS dose (NaHS, 1-1000 µM)-dependently potentiated BK currents and open probability. Co-incubation with a Ca blocker nifedipine (5 µM) or a chelator (ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 5 mM) did not alter HS-potentiated BK currents and open probability. NaHS also dose-dependently relaxed phenylephrine pre-constricted freshly prepared human UA rings, which was inhibited by IBTX. Thus, HS stimulated human UA relaxation at least partially via activating SM BK channels independent of extracellular Ca.