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The host defense system employs elaborate mechanisms to combat invading viruses. Here, we demonstrate that tripartite motif containing 45 (TRIM45) restricts the replication of different subtypes of influenza virus. TRIM45 interacted with and reduced the level of viral polymerase basic protein 2 (PB2). PB2 associated with heat shock cognate protein 70 (HSC70) and lysosomal-associated membrane protein type 2A (LAMP-2A), and was directed for lysosomal degradation via chaperone-mediated autophagy (CMA). TRIM45 promoted LAMP-2A expression and enhanced PB2/LAMP-2A binding, thereby facilitating CMA-dependent PB2 degradation. Mechanistically, TRIM45 employed its E3 ubiquitin ligase activity to mediate the K48-linked polyubiquitination and proteasomal degradation of Ca2 + -dependent cysteine protease calpain 1 (CAPN1), which prevented CAPN1-mediated cleavage of LAMP-2A. Sequence analysis identified a highly conserved QMRDV motif at position 602–606 of PB2, which was required for its binding with LAMP-2A or HSC70. Strikingly, mutations of this motif abolished this binding and the degradation effect of TRIM45 on PB2, and a PB2-Q602A mutant virus exhibited increased replication and enhanced pathogenicity in mice. Collectively, our findings reveal that TRIM45 restricts influenza virus infection by promoting the degradation of viral PB2 protein via CMA.