Normal expression of the Wnt/β-catenin signal pathway plays an important role in regulation of normal cell growth and differentiation, and research on target sites in the Wnt/β-catenin signal pathway thus has become a hotspot on targeted anticancer therapy. Low-level laser irradiation (LLLI) was discussed for its influence on RKO colorectal cancer cells through the Wnt/β-catenin signal pathway. RKO cells were irradiated by the 632.8 nm infrared laser, and RT-PCR method was adopted for screening gene expressions of APC、β-catenin and DVL in the Wnt/β-catenin signal pathway of RKO cells irradiated by a gradient low-level laser. Results of the RT-PCR showed that the low-level laser with an energy density of 15?J/cm2 reduced β-catenin expression of RKO cells, and increased APC and DVL expressions, indicating both an increasing trend as the rising energy density and a statistical discrepancy. Related indicators of RKO cells were tested for their changes by qRT-PCR and Western blot methods, under the low-level laser irradiation of 15 J/cm2. Results of qRT-PCR showed that gene expressions of Wnt5A, LRP5 and TCF-4 indicated different levels of reduction, with a statistical discrepancy. Results of Western blot showed that reduction in protein expression of β-catenin, relative to that of the blank control group, had a statistically significant discrepancy. Both qRT-PCR and Western blot showed that low-level laser irradiation of 15 J/cm2 for short time still had an inhibiting effect on overexpression of Wnt/β-catenin signal pathway of RKO cells. By observation on low-level laser irradiation of 15 J/cm2 through viable and dead-cell kits staining, it can be seen that the dead RKO cells in experimental group were more than that in control group. In conclusion, low-level laser irradiation of 15 J/cm2 can delay proliferation of RKO colorectal cancer cells through inhibition on overexpression of Wnt/β-catenin signal pathway, and is dose-dependent with a time-accumulation effect; it can also activate the apoptosis pathway and therefore incur cell apoptosis. This paper, in view of the above, provided a new thinking for the treatment of colorectal cancer.