Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.