UNLABELLED: Antiviral effectors and cytokines are critical components of host innate immunity. However, the regulatory mechanisms governing the roles of these molecules in host-virus interactions are still unclear. Although long non-coding RNAs (lncRNAs) have been recognized as key players in various biological processes, their involvement in the complement system of host antiviral defenses remains to be explored. In this study, we discovered a novel, unannotated lncRNA, called DARVR. DARVR was found to be an intergenic lncRNA and inhibited rotavirus (RV) replication in MA104 cells. Mechanistically, we found that complement 3 (C3) was upregulated following RV infection in a LAMB1-dependent manner. However, LAMB1 expression was downregulated by miR-365-1-5p, resulting in the inhibition of the C3-mediated antiviral reaction. However, DARVR functioned as a competing endogenous RNA against miR-365-1-5p, promoting the expression of LAMB1 and thereby enhancing C3 activity and inhibiting RV replication. These results not only provide evidence demonstrating the involvement of lncRNAs in the regulation of RV infection but also highlight the role of complement factors in host innate immunity. IMPORTANCE: Long non-coding RNAs (lncRNAs) play versatile and critical roles in host-virus interactions, offering significant potential for developing targeted therapies to prevent or treat viral infections. Despite their importance, the involvement of lncRNAs in rotavirus infection remains underexplored. This study identifies a novel lncRNA that enhances complement factor C3 activity through the competing endogenous RNA (ceRNA) mechanism, effectively inhibiting rotavirus replication across different subtypes. These findings underscore the complex molecular interplay regulating complement factor activity during rotavirus infection and provide valuable insights into the host's antiviral mechanisms. This research paves the way for innovative therapeutic strategies targeting lncRNAs and complement factors to combat viral infections more effectively.