Coxsackievirus B5 (CVB5) is a major causative agent of hand, foot, and mouth disease (HFMD) and is also linked to severe central nervous system complications and even death. Unfortunately, no specific antiviral drugs and preventive vaccines are currently available. Long non-coding RNAs (lncRNAs) are believed to play significant roles in various diseases. In our research, we identified a novel lncRNA, LINC2781, which shows significantly increased expression after CVB5 infects SH-SY5Y cells. Characteristic analysis reveals that the expression of LINC2781 is time- and dose-dependent upon CVB5 infection and is highly expressed in the intestines and spleen of CVB5-infected mice. Functional studies have demonstrated that LINC2781 activates the JAK-STAT pathway via STAT1 and promotes the expression of IFN-stimulated genes (ISGs), thereby inhibiting CVB5 replication. Mechanistically, LINC2781 directly binds to GTPase-activating protein SH3 domain-binding protein 2 (G3BP2) and blocks the G3BP2-STAT1 interaction, thus preventing G3BP2 from degrading STAT1 through ubiquitination. In vivo, LINC2781 decreases the susceptibility of BALB/c mice to viral infection and reduces lesions in the intestines and spleen caused by virus. Our results demonstrate that CVB5-inducible LINC2781 strengthens STAT1 activation by removing the suppressive effects of G3BP2 in immune responses, providing a foundation for developing a drug based on function of the lncRNA.