Feline calicivirus (FCV), causing respiratory disease in cats, contains a positive-sense single-stranded RNA genome of approximately 7.7 kb. Currently, there is no effective treatment for FCV infection. Interferon lambda (IFN-λ) is crucial for the innate immune response in epithelial cells. IFN-λ can exert antiviral activities by triggering specific sets of IFN-stimulated genes (ISGs). In this study, recombinant feline interferon lambda 1 (rFeIFN-λ1) was cloned and overexpressed in Escherichia coli and purified to demonstrate its antiviral activity against FCV in Crandell–Rees feline kidney cells. rFeIFN-λ1 was non-cytotoxic and displayed a dose-dependent induction of several antiviral protein genes, including ISG15, Mx1, PKR, and OAS1. rFeIFN-λ1 significantly suppressed FCV replication when the cells were treated 1 day prior to viral infection. Suppression of viral replication was confirmed using RT-qPCR, western blotting, plaque assays, and immunofluorescence assays. To our knowledge, this is the first in vitro study showing that rFeIFN-λ1 effectively inhibits FCV replication in feline cells. These results indicate that rFeIFN-λ1 may be clinically useful as a novel immunostimulatory antiviral agent for the treatment of FCV infection.