Recent studies have suggested that the dysregulation of microRNAs (miRNAs) plays a critical role in the progression of human cancers， including gastric cancer (GC). miR-143 had been reported to function as a tumor suppressor in GC. However， the exact molecular mechanism of how miR-143 participates in GC progression remains to be determined. In this present study， we revealed that the expression of miR-143 was significantly downregulated in human GC tissues and cell lines compared with normal tissues and a normal gastric epithelium cell line. In addition， upregulation of the expression of miR-143 in a GC cell line inhibited cell proliferation and induced cell cycle arrested in the G0/G1 phase. Furthermore， GATA6 was identified as a direct target of miR-143 in GC using the luciferase reporter assay. Upregulation of miR-143 inhibited the expression of GATA6 in GC cell lines. Moreover， the overexpression of GATA6 could attenuate the effect of miR-143 on cell proliferation in the GC cell lines. Collectively， these data indicated that miR-143 plays a tumor suppressor role partly through regulating the expression of GATA6 in GC. Therefore， targeting miR-143 may be a novel therapeutic method for GC.