Infectious pancreatic necrosis virus (IPNV) is the pathogen of infectious pancreatic necrosis (IPN), which can cause high mortality in salmonids, harm the healthy development of salmon-trout aquaculture, and lead to huge economic losses. However, in China, there is currently neither a commercially available vaccine to prevent IPNV infection nor antiviral drugs to treat IPNV infection. The genome of IPNV consists of two segments of dsRNA named A and B. Segment B encodes the RNA-dependent RNA-polymerase (RdRp) VP1 which is essential for viral RNA replication and is therefore considered an important target for the development of antiviral drugs. In this study, we investigate whether 2′-C-methylcytidine (2CMC), a nucleoside analog which target viral polymerases, has an inhibitory effect on IPNV both in vitro and in vivo. The results show that 2CMC inhibits IPNV infection by inhibiting viral RNA replication rather than viral internalization or attachment. In vivo experiment results showed that 2CMC could inhibit viral RNA replication and reduce viral load in rainbow trout (Oncorhynchus mykiss). In our study, we have revealed that 2CMC has a potent inhibitory effect against IPNV infection. Our data suggest that 2CMC is an attractive anti-IPNV drug candidate which will be highly valuable for the development of potential therapeutics for IPNV.