Lymphocyte chemotaxis plays important roles in immunological reactions, although the mechanism of its regulation is still unclear. We found that the cytosolic Na(+)-dependent mitochondrial Ca(2+) efflux transporter, NCLX, regulates B lymphocyte chemotaxis. Inhibiting or silencing NCLX in A20 and DT40 B lymphocytes markedly increased random migration and suppressed the chemotactic response to CXCL12. In contrast to control cells, cytosolic Ca(2+) was higher and was not increased further by CXCL12 in NCLX-knockdown A20 B lymphocytes. Chelating intracellular Ca(2+) with BAPTA-AM disturbed CXCL12-induced chemotaxis, suggesting that modulation of cytosolic Ca(2+) via NCLX, and thereby Rac1 activation and F-actin polymerization, is essential for B lymphocyte motility and chemotaxis. Mitochondrial polarization, which is necessary for directional movement, was unaltered in NCLX-knockdown cells, although CXCL12 application failed to induce enhancement of mitochondrial polarization, in contrast to control cells. Mouse spleen B lymphocytes were similar to the cell lines, in that pharmacological inhibition of NCLX by CGP-37157 diminished CXCL12-induced chemotaxis. Unexpectedly, spleen T lymphocyte chemotaxis was unaffected by CGP-37157 treatment, indicating that NCLX-mediated regulation of chemotaxis is B lymphocyte-specific, and mitochondria-endoplasmic reticulum Ca(2+) dynamics are more important in B lymphocytes than in T lymphocytes. We conclude that NCLX is pivotal for B lymphocyte motility and chemotaxis.