Retinoic acid receptor alpha (RARalpha) plays an important role in mediating all-trans retinoic acid (ATRA) signals. In this study, we found that ATRA up-regulated RARalpha mRNA and protein expression in gastric cancer BGC-823 cells. However, in breast cancer MCF-7 cells it down-regulated RARalpha protein expression with no effect on its RARalpha mRNA. Immunoprecipitation/Western blot analysis showed that, although sumoylated and ubiquitinated RARalpha existed simultaneously in both cancer cell lines, ATRA exerted different regulatory effects on sumoylation and ubiquitination of RARalpha. In MCF-7 cells, ATRA treatment enhanced the ubiquitination of RARalpha and the subsequent degradation of RARalpha through the ubiquitin/proteasome pathway. This resulted in a reduction in the DNA binding activity of RARalpha/retinoid X receptor alpha (RXRalpha) heterodimer, the separation of RXRalpha from RARalpha and the translocation of RXRalpha from the nucleus to the cytoplasm. By contrast, in BGC-823 cells, ATRA augmented sumoylation, not ubiquitination, of RARalpha. The stability of sumoylated RARalpha was significantly stronger than in non-sumoylated RARalpha. These results also showed an increase in the DNA binding activity of the RARalpha/RXRalpha heterodimer and the stability of nuclear localization of this heterodimer, which normally facilitates the ATRA signal transduction. In conclusion, our results reveal a novel mechanism for the regulation of RARalpha-dependent signal transduction through the ubiquitin/proteasome pathway in breast cancer cells and the sumoylation pathway in gastric cancer cells.