Background <p>Rotavirus (RV) infection damages mature intestinal epithelial cells and is a leading cause of severe diarrhea in infants and young animals. However, the host cell death pathways involved in RV infection remain incompletely understood. This study used an RV-infected IPEC-J2 cell model and integrated transcriptomic, proteomic, and functional assays to investigate whether RV infection is associated with ferroptosis and NCOA4-mediated ferritinophagy.</p> Results <p>High-throughput sequencing and data-independent acquisition (DIA)-based quantitative proteomic analysis showed enrichment of ferroptosis, autophagy, and cell death-related signaling pathways after RV infection. Functional assays showed that RV infection was associated with intracellular Fe²⁺ accumulation and lipid peroxidation. The ferroptosis inducer (erastin) increased VP6 expression, whereas the ferroptosis inhibitor (Fer-1) attenuated this effect. NCOA4, a ferritinophagy receptor, was upregulated after RV infection. NCOA4 knockdown reduced RV-associated Fe²⁺ accumulation, ROS production, ferritin degradation-related changes, and VP6 expression, whereas NCOA4 overexpression enhanced these responses. The autophagy inhibitor (3-MA) and the lysosomal blocker (CQ) attenuated RV-associated ferroptosis markers and VP6 expression, consistent with involvement of the autophagy-lysosome pathway in ferritin turnover.</p> Conclusions <p>These findings support a model in which RV infection promotes ferroptosis-associated changes and VP6 expression through NCOA4-mediated ferritinophagy in IPEC-J2 cells. As this study was performed in vitro and did not directly measure epithelial transport, barrier function, infectious viral yield, or diarrheal outcomes, further in vivo studies are needed to determine the contribution of this pathway to RV pathogenesis.</p>