miR-101-3p has been identified as a tumor suppressor in several cancers， but its exact role in gastric adenocarcinoma is still largely unknown. In this study， we found that， compared with the RGM-1 human normal gastric epithelial cells， miR-101-3p was significantly downregulated in all six human gastric adenocarcinoma cell lines， including BGC-823， MNK-45， MGC-803， SGC-7901， AGS， and HGC-27. Overexpression of miR-101-3p suppressed both the proliferation and invasion of AGS gastric adenocarcinoma cells， and knockdown of miR-101-3p displayed the opposite effect. In addition， miR-101-3p could directly target and suppress the expression of the serum response factor (SRF) gene， which is a transcription factor of HOTAIR， a well-characterized tumor promoter lncRNA. miR-101-3p negatively regulated SRF-mediated transcription of HOTAIR. Moreover， silencing of either SRF or HOTAIR could counteract the promotion of gastric adenocarcinoma cell proliferation and invasion by miR-101-3p inhibition. Our findings indicate that miR-101-3p suppresses HOTAIR-induced proliferation and invasion through directly targeting SRF in gastric carcinoma cells.