We recently reported that an N-terminally truncated retinoid X receptor-α (tRXRα) produced in cancer cells acts to promote cancer cell growth and survival through AKT activation. However, how RXRα is cleaved and how the cleavage is regulated in cancer cells remain undefined. In this study, we demonstrated that calpain II could cleave RXRα protein in vitro, generating two truncated RXRα products. The cleavage sites in RXRα were mapped by Edman N-terminal sequencing to Gly(90)↓Ser(91) and Lys(118)↓Val(119). Transfection of the resulting cleavage product RXRα/90, but not RXRα/118, resulted in activation of AKT in cancer cells, similar to the effect of tRXRα. In support of the role of calpain II in cancer cells, transfection of calpain II expression vector or its activation by ionomycin enhanced the production of tRXRα, whereas treatment of cells with calpain inhibitors reduced the levels of tRXRα. Co-immunoprecipitation assays also showed an interaction between calpain II and RXRα. In studying the regulation of tRXRα production, we observed that treatment of cells with lithium chloride or knockdown of glycogen synthase kinase-3β (GSK-3β) significantly increased the production of tRXRα. Conversely, overexpression of GSK-3β reduced tRXRα expression. Furthermore, we found that the inhibitory effect of GSK-3β on tRXRα production was due to its suppression of calpain II expression. Taken together, our data demonstrate that GSK-3β plays an important role in regulating tRXRα production by calpain II in cancer cells, providing new insights into the development of new strategies and agents for the prevention and treatment of tRXRα-related cancers.