Natural killer (NK) cells have pivotal role in immunotherapy of human ovarian cancer (OC). AlthoughmicroRNAs (miRNAs) participate in dysfunction of NK cells, how and whether miR-140-3p regulates cytotoxicityof NK cells in OC are uncertain. miR-140-3p and mitogen activated protein kinase 1 (MAPK1)abundances were examined via quantitative real-time polymerase chain reaction or western blot. Tumornecrosis factor-a (TNF-alpha) and interferon-c (IFN-gamma) abundances were examined via enzyme linkedimmunosorbent assay. NK cytotoxicity to OC was evaluated via lactate dehydrogenase release. The relevanceof miR-140-3p and MAPK1 was proved via luciferase activity analysis. Murine xenograft experiment wasapplied to assess the function of miR-140-3p on NK cytotoxicity. miR-140-3p was elevated and MAPK1 wasdeclined in NK cells from OC patients, while the levels were reversed after treatment of interleukin-2 (IL-2).MiR-140-3p addition mitigated IFN-gamma and TNF-alpha production induced via IL-2 as well as NK-92 cytotoxicityto OC cells. Additionally, MAPK1 was negatively regulated via miR-140-3p and ablated the influence of miR-140-3p on cytotoxicity, cytokines levels. Besides, miR-140-3p enrichment facilitated tumor growth via suppressingfunction of NK cells in a xenograft model. miR-140-3p suppressed NK cytotoxicity to OC cells viamediating MAPK1, indicating a new avenue of ameliorating NK cells function for OC treatment.