Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5-/- mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5+/+ and Slc1a5-/- bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5-/- bone marrow cells was reduced to 70% of that of cells from Slc1a5+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5+/+ or Slc1a5-/- mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5-/- mice was severely impaired compared with those from Slc1a5+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5-/- osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.