Objective To investigate the effect of knockdown of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) on cell proliferation, cell cycle and apoptosis of MGC-803 human gastric cancer cells in vitro. Methods The TCGA database was downloaded from UCSC Cancer Browser and to search for the differential expressions of YTHDF2 mRNA in gastric cancer tissues. Short hairpin RNA (shRNA) targeting YTHDF2 was designed and cloned into lentivirus expression vector pLKO.1. Furthermore, MGC-803 gastric cancer cells were transfected with pLKO.1-shRNA to knockdown the expression of YTHDF2, which was confirmed by the detection of YTHDF2 mRNA and protein expression using real-time quantitative PCR and Western blotting, respectively. Then cell proliferation was observed by CCK-8 assay, and cell cycle and apoptosis were examined by flow cytometry. Results According to the TCGA database, the expression of YTHDF2 mRNA in gastric cancer was significantly higher than that in the normal tissues. MGC-803 stably expressing YTHDF2-shRNA was successfully established. Furthermore, the proliferation capacity of YTHDF2-shRNA-expressing MGC-803 cells was significantly inhibited compared with the controls. Similarly, the percentage of YTHDF2-shRNA-expressing MGC-803 cells in G1 phase increased and in S phase decreased compared with the controls. Meanwhile, apoptosis ratio of YTHDF2-shRNA-expressing MGC-803 cells was significantly higher compared with the control groups. Conclusion Knockdown of YTHDF2 in MGC-803 cells inhibits cell proliferation and promotes apoptosis.