The final assembly of rotavirus particles takes place in the endoplasmic reticulum (ER). In this work, we evaluated by RNA interference the relevance to rotavirus assembly and infectivity of grp78, protein disulfide isomerase (PDI), grp94, calnexin, calreticulin, and ERp57, members of the two ER folding systems described herein. Silencing the expression of grp94 and Erp57 had no effect on rotavirus infectivity, while knocking down the expression of any of the other four chaperons caused a reduction in the yield of infectious virus of about 50%. In grp78-silenced cells, the maturation of the oligosaccharide chains of NSP4 was retarded. In cells with reduced levels of calnexin, the oxidative folding of VP7 was impaired and the trimming of NSP4 was accelerated, and in calreticulin-silenced cells, the formation of disulfide bonds of VP7 was also accelerated. The knockdown of PDI impaired the formation and/or rearrangement of the VP7 disulfide bonds. All these conditions also affected the correct assembly of virus particles, since compared with virions from control cells, they showed an altered susceptibility to EGTA and heat treatments, a decreased specific infectivity, and a diminished reactivity to VP7 with monoclonal antibody M60, which recognizes only this protein when its disulfide bonds have been correctly formed. In the case of grp78-silenced cells, the virus produced bound less efficiently to MA104 cells than virus obtained from control cells. All these results suggest that these chaperones are involved in the quality control of rotavirus morphogenesis. The complexity of the steps of rotavirus assembly that occur in the ER provide a useful model for studying the organization and operation of the complex network of chaperones involved in maintaining the quality control of this organelle.