Simple SummaryRottlerin, a type of natural extract, can inhibit the activity of various viruses. Feline coronavirus (FCoV) causes a devastating disease in cats. In this study, we investigated whether rottlerin has inhibitory effects on FCoV. We demonstrated that rottlerin inhibits FCoV replication and FCoV-induced PKCδ phosphorylation. We showed that rottlerin affects the early (endocytosis) and late (syncytium formation) stages of FCoV replication. We also observed that the liposome encapsulation technique overcomes the drawbacks of rottlerin and enhances its efficacy. Therefore, we suggest that rottlerin-liposome is worth further investigation as a potential treatment for FCoV.AbstractRottlerin (R) is a natural extract fromMallotus philippensiswith antiviral properties. Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) that is characterized by systemic granulomatous inflammation and high mortality. We investigated the antiviral effect of liposome-loaded R, i.e., rottlerin-liposome (RL), against FCoV. We demonstrated that RL inhibited FCoV replication in a dose-dependent manner, not only in the early endocytosis stage but also in the late stage of replication. RL resolved the low solubility issue of rottlerin and improved its inhibition efficacy at the cellular level. Based on these findings, we suggest that RL is worth further investigation as a potential treatment for FCoV.