To clarify the role of mitochondrial Na-Ca exchange (NCXm) on antigen-receptor (BCR)-mediated Ca2+ signaling of B lymphocytes, we first carried out mathematical modeling of BCR-mediated Ca2+ signaling. Our computer simulation suggested that NCXm modulates both endoplasmic reticulum (ER) Ca2+ content and BCR-mediated cytoplasmic Ca2+ (Ca2+i) rise. The model predictions were validated by following experiments with DT40 B lymphocytes whose NCXm gene (NCLX) was knocked-out (NCLX+/-). In NCLX+/- cells, expression of NCLX protein was markedly reduced and cytoplasmic Na+-dependent Ca2+m efflux were significantly attenuated. ER Ca2+ content, which was estimated as ionomycin-induced Ca2+i increase in the nominally Ca2+-free solution, was reduced to about 21% of control cells (WT). ER Ca2+ uptake via Ca2+ pump (SERCA) was decreased by about 42% in NCLX+/- cells, while it was comparable to WT when mitochondria functions were suppressed. No significant difference was observed in content of SERCA mRNA. In consequence, BCR stimulation by anti-IgM antibody hardly induced Ca2+i rise in NCLX+/- cells. Inhibition of NCXm by CGP-37157 induced similar results in WT. Essentially the same results were obtained by silencing NCLX in A 20 B lymphocytes with small interfering RNA. Taken together, we concluded that NCXm supports ER Ca2+ filling and pivotal in BCR-mediated Ca2+ signaling in B lymphocytes.