The central nervous system (CNS) pericytes play an important role in brain microcirculation. Na⁺/H⁺ exchanger isoform 1 (NHE1) has been suggested to regulate the proliferation of non-vascular cells through the regulation of intracellular pH, Na⁺ and cell volume; however, the relationship between NHE1 and intracellular Ca²⁺, an essential signal of cell growth, is still not known. The aim of the present study was to elucidate the role of NHE1 in Ca²⁺ signaling and the proliferation of human CNS pericytes. The intracellular Ca²⁺ concentration was measured by fura-2 in cultured human CNS pericytes. The cells showed spontaneous Ca²⁺ oscillation under quasi-physiological ionic conditions. A decrease in extracellular pH or Na⁺ evoked transient Ca²⁺ rise followed by Ca²⁺ oscillation, whereas an increase in pH or Na⁺ did not induce the Ca²⁺ responses. The Ca²⁺ oscillation was inhibited by an inhibitor of NHE dose-dependently and by knockdown of NHE1 using RNA interference. The Ca²⁺ oscillation was completely abolished by thapsigargin. The proliferation of pericytes was attenuated by inhibition of NHE1. These results demonstrate that NHE1 regulates Ca²⁺ signaling via the modulation of Ca²⁺ release from the endoplasmic reticulum, thus contributing to the regulation of proliferation in CNS pericytes.