BACKGROUND Fungal immunomodulatory proteins (FIPs) are a newly identified protein family that exhibits immunomodulation and antitumor activities. However, little is known about the FIPs from Gomphidiaceae species. In this study, a novel FIP gene termed FIP‐cru was cloned from Chroogomphis rutilus, analyzed, and functionally expressed in Pichia pastoris GS115. In addtion, the bioactivity of rFIP‐cru was examined including hemagglutination, cytotoxicity, and interleukin‐2 assays in vitro. RESULTS FIP‐cru comprised 342 bp and encoded a peptide of 113 amino acids, which shared a high homology to other FIPs. rFIP‐cru was obtained by expressing the recombinant gene in P. pastoris GS115, with a yield of 148.5 mg L−1, and then purified via ammonium sulfate precipitation and agarose nickel magnetic nanobeads. The bioactivity assay showed that rFIP‐cru can agglutinate mouse and sheep red blood cells but not human cells. Additionally, rFIP‐cru can stimulate the proliferation of murine splenocytes and enhance the secretion of interleukin‐2. CONCLUSION FIP‐cru is a novel fungal immunomodulatory protein. The use of P. pastoris as an expression system will provide a feasible production method to obtain large amounts of rFIP‐cru to study its potential application in therapeutic and medical studies. © 2016 Society of Chemical Industry