Leukocyte cell-derived chemotaxin 2 (LECT2) is reported to be an immunorelevant protein in ayu (Plecoglossus altivelis). In this study, ayu LECT2 mature peptide (aLECT2m) was expressed as insoluble inclusion bodies in Escherichia coli. The denatured recombinant aLECT2m (raLECT2m) was refolded by a size-exclusion chromatography refolding process achieved by using arginine-containing mobile phase and a decreasing urea gradient. The in vitro chemotactic activity assay showed that the refolded raLECT2m had the bioactivity. By using suppression subtractive hybridization (SSH) method, we further identified up-regulated genes in ayu macrophages treated with refolded raLECT2m. These genes were tightly involved in endocytosis, hydrolysis, transcriptional regulation, signal transduction, and so on. Moreover, real-time quantitative PCR (RT-qPCR) results confirmed that selected 10 genes expression was significantly up-regulated in refolded raLECT2m-treated ayu macrophages. This study provides a basis for further studies of the mechanism of cytokine LECT2 in fish immune responses.