Zinc finger proteins (ZNFs) are a class of proteins widely distributed in the human genome, which have been found to play a role in the regulation of gene transcription and the occurrence and development of gastric cancer (GC). ZNF139 was found to be associated with GC in our previous experiments. The present study aimed to analyse the differences in ZNF139 protein expression in SGC7901 GC cells and in situ grafted GC tumors in nude mice prior to and following RNA interference inhibition, and to investigate the mechanisms underlying ZNF139 involvement in the occurrence, development and chemosensitivity of GC. A ZNF139-targeted small interfering (si) RNA plasmid was constructed and transfected into the cancer cells and in situ grafted tumors. The MTT assay was used to investigate the alterations in chemosensitivity prior to and following transfection of siRNA-ZNF139. The two-dimensional difference gel electrophoresis and liquid chromatography-mass spectrometry techniques were used to identify the different protein points prior to and following siRNA-ZNF139 transfection. Western blot analysis was performed to confirm the identified proteins. In the siRNA-ZNF139 group, the growth of the cancer cells and in situ grafted tumors significantly decreased. However, the post-interference chemosensitivity to 5-fluorouracil, cisplatin and mitomycin C significantly increased. In the in vivo and in vitro experiments, the expression of pyridoxal kinase (PDXK) was upregulated, whereas the expression levels of annexin A2 (ANXA2) and fascin were downregulated following transfection. Western blot analysis confirmed the results for PDXK, ANXA2 and fascin by proteomics. Therefore, ZNF139 may participate in the occurrence, development and chemosensitivity of GC by promoting the expression of ANXA2 and fascin, while inhibiting the expression of PDXK.