Objective It remains a controversy whether 5-lipoxygenase (5-LOX) is associated with colon cancer stem cells.This study was to investigate the effect of the 5-LOX inhibitor MK886 in maintaining the stemness of the human colon cancer cell line HT-29.Methods Using CCK-8 assay, we examined the inhibitory effects of different concentrations of MK886 (12.5, 25, 50, 75, 100, and 200 μmol/L) on the colon cancer HT-29 cells cultured in vitro and calculated its half-inhibitory concentration (IC50).Then, we detected the effects of MK886 IC50 on the clone-and sphere-forming abilities of the cells, determined the mRNA expressions of the stemness markers CD133, Lgr5, Oct4 and Ascl2 by real-time PCR after 24 and 48 hours of MK886 IC50 intervention, and measured their protein expressions by Western blotting after 24, 48 and 72 hours of MK886 IC50 intervention.Results The inhibition rates of MK886 on the HT-29 cells at 24 and 48 hours were significantly increased in a time-and dose-dependent manner ([14.99±3.06] and [19.98±0.57]％ at 12.5 μmol/L, [20.46±1.14] and [34.97±6.02]％ at 25 μmol/L, [50.76±5.94] and [66.90±5.74]％ at 50 μmol/L, [66.84±1.77] and [73.11±2.48]％ at 75 μmol/L, [72.67±2.36] and [77.78±3.30]％ at 100 μmol/L, [83.67±0.24] and [84.69±2.24] ％ at 200 μmol/L) as compared with the blank control (0％ and 0％) (P<0.05).The clone-forming rate and number of spheres formed were remarkably lower in the MK886 intervention than in the control group ([10.60±1.71] vs [44.67±3.21]％, P<0.05;6.00±1.60 vs 19.07±2.89, P<0.05).After 24 and 48 hours of MK886 intervention, the mRNA expression of CD133 in the HT-29 cells was markedly up-regulated in comparison with that at 0 hour (0.72±0.10 and 0.39±0.07 vs 1.66±0.33, P<0.05), and so were those of Lgr5, Oct4 and Ascl2 (P<0.05).Conclusion The 5-LOX inhibitor MK886 can inhibit the proliferation and clone-and sphere-forming abilities of human colon cancer HT-29 cells by down-regulating the expressions of the stemness markers and thus suppressing the stemness of the colon cancer stem cells.