Micro-RNA-21 (miR-21) is a vital regulator of colorectal cancer (CRC) progression and has emerged as a potential therapeutic target in CRC treatment. Our study using real-time PCR assay found that a secondary bile acid, lithocholic acid (LCA), stimulated the expression of miR21 in the CRC cell lines. Promoter activity assay showed that LCA strongly stimulated miR21 promoter activity in HCT116 cells in a time- and dose-dependent manner. Studies of chemical inhibitors and miR21 promoter mutants indicated that Erk1/2 signaling, AP-1 transcription factor, and STAT3 are major signals involved in the mechanism of LCA-induced miR21 in HCT116 cells. The elevation of miR21 expression was upstream of the phosphatase and tensin homolog (PTEN) inhibition, and CRC cell proliferation enhancement that was shown to be possibly mediated by PI3K/AKT signaling activation. This study is the first to report that LCA affects miR21 expression in CRC cells, providing us with a better understanding of the cancer-promoting mechanism of bile acids that have been described as the very first promoters of CRC progression.