Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggested that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic RAR and RXR receptors. RA did not affect the levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CIMPR and transiently transfected GFP and RFP full-length CIMPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CIMPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CIMPR. Transient CIMPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA. Our results suggest that RA induces AUT acidification and maturation, a process critical in the cellular autophagic mechanism.