Objective To explore the regulation roles of microRNA-1285 (miR-1285) and its target DDX3X in Senecavirus A (SVA) infected PK-15 cells.Method By qRT-PCR, double luciferase activity and Western blot, the effects of miR-1285 and its target DDX3X on IFN-β secretion and the RIG-I signaling pathway were studied, and their effects on the expression of SVA 3C protein gene were analyzed.Result In SVA infected PK-15 cells, the expression of miR-1285 increased significantly, and there was a negative targeting relationship between miR-1285 and DDX3X. Both miR-1285 and DDX3X promoted the transcription and protein expression of IFN-β. MiR-1285 regulated MAVS and TRAF3 signaling molecules in the RIG-I signaling pathway by targeting DDX3X. For SVA 3C protein, DDX3X significantly inhibited the transcription of 3C and reversed the up-regulation trend induced by miR-1285.Conclusion After infecting PK-15 cells with SVA, host miR-1285 and its target DDX3X can regulate the expression of IFN-β and the viral 3C protein, which will lay a foundation for clarifying the molecular mechanism of miRNAs regulating SVA infection, and provide a new scientific basis for the prevention, control and diagnosis of SVA.