Tripartite motif protein 21 (TRIM21) is an E3 ubiquitin ligase and cytosolic antibody receptor of the TRIM family. Previous reports have indicated that TRIM21 plays an important role during viral infection. This study aimed at examining the role of TRIM21 in the replication of porcine epidemic diarrhea virus (PEDV) and showed that TRIM21 inhibits PEDV proliferation by targeting and degrading the nucleocapsid (N) protein through the proteasomal pathway. Furthermore, the endogenous expression of TRIM21 was found to be downregulated by PEDV infection in Vero and LLC-PK1 cells. Overexpression of TRIM21 inhibited PEDV replication, whereas knockdown of TRIM21 increased viral titers and N protein levels. TRIM21 was found to interact and colocalize with the N protein, and the TRIM21-mediated antiviral effect was dependent on its ubiquitin ligase activity, which engages in polyubiquitination and degradation of the N protein in a proteasome-dependent manner. Taken together, these findings provide information about the role of TRIM21 in PEDV proliferation and increase our understanding of host-virus interactions.