Replication of peste des petits ruminants virus (PPRV) strongly depends on the cellular environment and resources of host cells including nucleoside pool. Thus, enzymes involved in nucleoside biosynthesis (such as pyrimidine biosynthesis pathway) are regarded as attractive targets for antiviral drug development. Here, we demonstrate that brequinar (BQR) and leflunomide (LFM) which are two specific inhibitors of DHODH enzyme and 6-azauracil (6-AU) which is an ODase enzyme inhibitor robustly inhibit PPRV replication in HEK293T cell line as well as in peripheral blood mononuclear cells isolated from goat. We further demonstrate that these agents exert anti-PPRV activity via the depletion of purimidine nucleotide. Interestingly, these inhibitors can trigger the transcription of antiviral interferon-stimulated genes (ISGs). However, the induction of ISGs is largely independent of the classical JAK-STAT pathway. Combination of BQR with interferons (IFNs) exerts enhanced ISG induction and anti-PPRV activity. Taken together, this study reveals an unconventional novel mechanism of crosstalk between nucleotide biosynthesis pathways and cellular antiviral immunity in inhibiting PPRV replication. In conclusion, targeting pyrimidine biosynthesis represents a potential strategy for developing antiviral strategies against PPRV.