Ganoderma sinensis fungal immunomodulatory protein (FIP-gsi) was a new member of FIPs family. Based on the cloning of FIP-gsi gene from G. sinensis, this paper reported that FIP-gsi gene was expressed in Escherichia coli expression system. Then, the recombinant proteins were analyzed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Finally, the immunomodulatory activity was examined by inducing cytokine genes expression. The results showed that the recombinant FIP-gsi protein could be expressed in E. coli and got the yield of about 25% of the soluble form in the total soluble protein. The FIP-gsi protein was composed of 111 amino acids, and the sequence of homologous rate was 88.6% with FIP-glu (LZ-8). Furthermore, it could enhance the levels of interleukin (IL)-2, IL-3, IL-4, interferon gamma, tumor necrosis factor alpha, and IL-2 receptor (IL-2R) in mouse spleen cells.