The retinoid X receptor alpha (RXRα) is an important therapeutic target impacting diverse biological processes. Activation of RXRα is known to suppress cancer cell growth. However, the cellular mechanism has been elusive. In the present study, we addressed its role during stem cell differentiation and the underlying connections with carcinogenesis. RXRα was significantly upregulated following the differentiation of human mesenchymal stem cell (hMSC) toward the formation of endothelial cell (EC). However, overexpression of RXRα in hMSC provoked a senescence-like phenotype accompanied by the elevation of tumor suppressor p53, p21, and p16. Consistently, RXRα level was suppressed in cancer cells (~five times lower compared to differentiated hMSC), and its elevation could inhibit the proliferation, migration, and angiogenesis of cancer cells. We further demonstrated that these inhibitory effects were related to RXRα's interaction with estrogen receptor α (ERα) as well as EGF and ANGPTL3 through modulating PI3K/AKT signaling pathway by AKT and FAK phosphorylation. Moreover, RXRα inhibited glycolytic metabolism in cancer cells, which might be underlying its inhibition of differentiation and carcinogenic features. These data suggest that RXRα acts as a suppressor rather than a driving force during stem cell differentiation, and unbalanced RXRα can trigger multiple yet connected signaling pathways in preventing carcinogenesis.