Abstract Multiple myeloma (MM) is an incurable hematologic malignancy. While recent therapies have significantly improved survival in MM patients, drug resistance and refractory phenomenon underscores the urgent need of new therapeutic targets. Methylenetetrahydrofolate dehydrogenase 2(MTHFD2) has been widely reported as a potential and promising anti-cancer target, but its role and underlying mechanisms remain unclear in MM. We aimed to investigate the biologic function and mechanisms of MTHFD2 in MM. First, we demonstrated that MTHFD2 is overexpressed in MM and associated with poor prognosis. We then illustrated that targeting MTHFD2 exhibits anti-MM effects in vitro and in vivo. Mechanistically, targeting MTHFD2 inhibited glycolysis and mitochondrial respiration in MM cells. For the nonmetabolic function of MTHFD2, we found that MTHFD2 knockdown affected the unfolded protein response (UPR) via decreasing expression of the splice form of X-box binding protein 1 (XBP1s). Importantly, the level of MTHFD2 in MM cells was associated with sensitivity of bortezomib, and targeting MTHFD2 synergizes with bortezomib against MM in vitro and in vivo. In summary, our innovative findings suggest that MTHFD2 is a promising target for MM, targeting it alters metabolic homeostasis of MM and synergizes with bortezomib to inhibit MM.