Senecavirus A (SVA) is a non-enveloped, positive sense, single-stranded RNA virus that causes vesicular diseases in pigs. Interferon-induced transmembrane 3 (IFITM3) is an interferon-stimulated gene (ISG) that exhibits broad antiviral activity. We investigated the role of IFITM3 in SVA replication. Both viral protein expression and supernatant virus titer were significantly increased when endogenous IFITM3 was knocked down by approximately 80% in human non-smallcell lung carcinoma cell line (NCI-H1299) compared to silencing RNA control. Interestingly, overexpression of exogenous IFITM3 in NCI-H1299 cells also significantly enhanced viral protein expression and virus titer compared to vector control, which was positively correlated with induction of autophagy mediated by IFITM3 overexpression. Overall, our results indicate an antiviral role of endogenous IFITM3 against SVA. The exact molecular mechanisms by which endogenous IFITM3 limits SVA replication remain to be determined in future studies.