Abstract: To construct the prokaryotic expression system of fatty acid binding protein 5（FABP5） gene mutants and evaluate the anti-prostate cancer activity of the mutants in vitro, site-directed mutation technique was used to mutate FABP5 protein’s three key sites of fatty acid binding, and a prokaryotic expression system was constructed to express, isolate and purify the recombinant protein. Cytotoxicity, scratch wound, and cell invasion tests were used to evaluate the effects of recombinant FABP5 mutant proteins on proliferation, migration, and invasion of 22 RV1 and PC3 prostate cancer cells. The results showed that the DNA after fixed-point mutation was linked to the expression vector pQE32 and transferred into Escherichia coli BL21（DE3）, the sequence was determined to be correct, the recombinant expressed engineering bacteria were constructed, and the recombinant protein induced by affinity chromatography was purified to a higher purity. The inhibition effect of the three mutants on cell proliferation, migration and invasion was more obvious than that of the three single mutants and three double mutants;There was little difference in cell proliferation, migration and invasion inhibition between single and double mutant groups. The wild-type recombinant protein promoted the proliferation, migration and invasion of the two cells.In this study, the three mutants of FABP5 protein screened out from all mutants had a good inhibitory effect on prostate cancer cells. This result provided a reference for the subsequent development of castration resistant prostate cancer（CRPC） protein drugs.