MicroRNAs (miRNAs) play important roles in tumorigenesis and metastasis. In this study, we investigated miR-200b expression in endometrial adenocarcinomas and normal adjacent tissues and found that miR-200b is more highly expressed in cancer tissues than in normal adjacent tissues. A novel target of miR-200b, tissue inhibitor of metalloproteinase 2 (TIMP2), was predicted using a bioinformatics approach and was confirmed in human endometrial cancer cell line HEC-1A cells by luciferase assay, quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. We found that miR-200b repressed TIMP2 expression at both the messenger RNA and protein levels, although a family member, miR-200a, had no such effect. Using reverse gelatin zymography, we showed that miR-200b enhances matrix metallopeptidase 2 (MMP2) activity by downregulating TIMP2 expression in HEC-1A cells. These data suggest that miR-200b may play an important role in the metastasis of endometrial adenocarcinomas.