Cancer chemotherapy with methotrexate (MTX) is known to cause bone loss. However, the underlying mechanisms remain unclear. This study investigated the potential role of MTX-induced pro-inflammatory cytokines and activation of NF-κB in the associated osteoclastogenesis in rats. MTX (0.75 mg/kg per day) was administered for 5 days, and bone and bone marrow specimens were collected on days 6, 9, and 14. Compared with a normal control, MTX increased the density of osteoclasts within the metaphyseal bone and the osteoclast formation potential of marrow cells on day 9. RT-PCR analysis of mRNA expression for pro-osteoclastogenic cytokines in the metaphysis indicated that, although the receptor activator of NF-κB ligand/osteoprotegerin axis was unaffected, expression of tumor necrosis factor (TNF)-α, IL-1, and IL-6 increased on day 9. Enzyme-linked immunosorbent assay analysis of plasma showed increased levels of TNF-α on day 6 and of IL-6 on day 14. Plasma from treated rats induced osteoclast formation from normal bone marrow cells, which was attenuated by a TNF-α-neutralizing antibody. Indicative of a role for NF-κB signaling, plasma on day 6 increased NF-κB activation in RAW 264.7 cells, and plasma-induced osteoclastogenesis was abolished in the presence of the NF-κB inhibitor, parthenolide. Our results demonstrate mechanisms for MTX-induced osteoclastogenesis and show that MTX induces osteoclast differentiation by generating a pro-osteoclastogenic environment in both bone and the circulation, specifically with increased TNF-α levels and activation of NF-κB.