Technical Abstract: Butyrate, a normal constituent of the colonic luminal contents, is produced by the bacterial fermentation of dietary fibers and resistant starches. It has been hypothesized that butyrate may inhibit the invasion of tumor cells. The purpose of the present study was to investigate the effects of prolonged butyrate treatment on the growth, migration, and invasion characteristics of HT1080 cells, a well defined human invasive cancer cell line. HT1080 cells cultured in the presence of 0.5 and 1 mmol/L butyrate for 14 days exhibited an increase in the G1 and G2 fractions with a concomitant drop in the S-phase, thus showed slower cell growth. Interestingly, both 0.5 and 1 mmol/L butyrate inhibited the migration rate (53% and 67%, respectively), and invasion rate (74% and 79%, respectively) of the tumor cells when compared with the untreated (control) cells. The protein and mRNA level of the tissue inhibitors of metalloproteinase-1 (TIMP-1) and TIMP-2 were significantly increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. Enzymatic activities and the mRNA level of the latent forms of matrix metalloproteinase (MMP), pro-MMP-2 and pro-MMP-9, were also increased in HT1080 cells cultured with 0.5 and 1 mmol/L butyrate. In contrast, the active form of MMP-2 was detectable by zymographic analysis in control but not butyrate conditioned media. Collectively, these results demonstrate that prolonged and low-dose butyrate treatment enhances both pro-metastasis MMP-2, -9 and anti-metastasis TIMP-1, -2 expression. However, the net effect is the inhibition of the active form of MMP-2. More importantly, the inhibitory effect of prolonged 0.5 mmol/L butyrate treatment on cell migration and invasion potential was far greater than its effect on cell growth.