Background: Intracellular pH (pHi) is a pivotal factor for cellular functions and homeostasis. Apart from passive intracellular buffering capacity, active transmembrane transporters responsible for kinetic changes of pHi impacts. Acid extrusion transporters such as Na+/H+ exchanger (NHE) and Na+/HCO3 − cotransporter (NBC) have been found to be activated when cells are in an acidic condition in different cell types. However, such far, the pHi regulators have not been characterized in human umbilical artery smooth muscle cells (HUASMCs). Materials and Methods: We, therefore, investigated the mechanism of pHi recovery from intracellular acidosis, induced by NH4Cl-prepulse, using pH-sensitive fluorescence dye: 2’,7’-bis(2-carboxethyl)-5(6)-carboxy-fluorescein in HUASMCs. Cultured HUASMCs were derived from the segments of the human umbilical artery that were obtained from women undergoing children delivery. Results: The resting pHi is 7.23 ± 0.03 when cells in HEPES (nominally HCO3 −-free) buffered solution. The resting pHi is higher as 7.27 ± 0.03 when cells in CO2/HCO3 −-buffered solution. In HEPES-buffered solution, a pHi recovery following induced intracellular acidosis could be inhibited completely by 30 μM HOE 694 (a specific NHE inhibitor) or by removing [Na+]o. In 5% CO2/HCO3 −-buffered solution, 30 μM HOE 694 slowed the pHi recovery from the induced intracellular acidosis only. On the contrary, HOE 694 adding together with 0.2 mM 4,4’-diisothiocyanatostilbene-2,2’-disulphonic acid (a specific NBC inhibitor) or removal of [Na+]o entirely blocked the acid extrusion. By using Western blot technique, we demonstrated that four different isoforms of NBC, that is, SLC4A8 (NBCBE), SLC4A7 (NBCn1), SLC4A5 (NBCe2) and SLC4A4 (NBCe1), co-exist in the HUASMCs. Conclusions: We demonstrate, for the 1st time, that apart from the housekeeping NHE1, another Na+ couple HCO3 −-transporter, that is, NBC, functionally coexists to responsible for acid-extruding in HUASMCs.