miRNAs are a class of endogenous non-coding RNA, which can regulate downstream target genes through binding to the 3'UTR of those genes. Numerous studies have indicated that abnormal expression of miRNAs is implicated in tumor development. Aberrant expression of miR-31 has been found in various cancers, including colorectal cancer. Here, we show that miR-31 is upregulated in human colon cancer tissues and cell lines, and that repression of miR-31 inhibited colon cancer cell proliferation and colony formation in soft agarose. To further elucidate the mechanism underlying the role of miR-31 in promoting colon cancer, we used online miRNA target prediction databases and found that the tumor suppressor RhoTBT1 may be a target of miR-31. Imunohistochemistry assay revealed that RhoBTB1 was significantly decreased in HT29 cells. In addition, ectopic expression of miR-31 reduced RhoBTB1 in the colon cancer cell line HT29. The results suggested that suppression of RhoBTB1 may be responsible for colon tumorigenesis, which was inhibited directly by miR-31. The results of MTT and soft agarose colony-formation assays showed that knockdown of RhoBTB1 by RNAi induced cell proliferation, and colony formation in soft agarose, which mimicked the function of miR-31. This further suggested that suppression of RhoBTB1 was responsible for colon tumorigenesis. In conclusion, we found that miR-31 acts as an oncogene in colon cancer and identified RhoBTB1 as a new target of miR-31 further study demonstrated that miR-31 contributed to the development of colon cancer at least partly by targeting RhoBTB1.