AML with Mt NPM1 has relatively good responses to induction therapy. However, a proportion of NPMc+ AML cells cannot be cleared by conventional treatments. Therefore, we determined the therapeutic efficacy of deguelin that has demonstrated extensive biological activity with low toxicity. We previously reported that deguelin selectively reduces Mt NPM1, as well as induces differentiation and potentiates apoptosis in NPMc+ AML cells. Nevertheless, little information is available regarding the mechanism of deguelin-induced differentiation. Here, we investigated the role of deguelin in the induction of NPMc+ AML cell differentiation. Deguelin at the nontoxic concentration of 2 μM strongly inhibited cell growth but reduced apoptosis in OCI-AML3 cells carrying Mt NPM1, whereas the antiproliferative effect was minimal in OCIM2 cells harboring Wt NPM1. Compared with OCIM2 cells that showed no response, deguelin-treated OCI-AML3 cells exhibited the morphological features of granulocytic/monocytic differentiation, increased expression of differentiation antigens, and a nitroblue tetrazolium reduction activity. Induction of differentiation was associated with downregulation of Mt NPM1 and SIRT1, but not Wt NPM1, which was accompanied by an increase in CEBPβ and G-CSFR expression, and further confirmed by sh-Mt NPM1 and sh-SIRT1. sh-Mt NPM1 treatment reduced SIRT1 expression, but did not change HDAC1/3 levels, suggesting that the decline of SIRT1 was partially accountable for the deguelin-induced, Mt-NPM1-related differentiation. Moreover, Mt NPM1 overexpression blocked deguelin-induced cell differentiation. Lastly, we showed that deguelin reduced the expression of Mt NPM1 via the ubiquitin-proteasome pathway. Taken together, our results suggest that deguelin may be a therapeutic candidate for NPMc+ AML.