Background and Aims The transforming growth factor β (TGF-β)/SMAD4 signaling pathway plays an important role in the occurrence and development of colorectal cancer. The expression of runt-related transcription factor 3 (RUNX3) was found to be remarkably decreased in colorectal tissues and it may exert tumor-suppressing effects. However, the relationship between the actions of RUNX3 and TGF-β/SMAD4 pathway has not yet been reported. Therefore, this study was conducted to investigate the expressions and effects of RUNX3 and SMAD4 in colorectal cancer tissue, and their mutual relationship.Methods A total of 98 paired samples of colorectal tissue and adjacent normal tissue were collected. The protein expressions of RUNX3 and SMAD4 were detected by Western blot, and the mRNA expression of SMAD4 was detected by qRT-PCR, respectively. The correlation between RUNX3 and SMAD4 expressions were analyzed. Human colorectal cancer SW480 cells were transfected with RUNX3 overexpression plasmid (RUNX3 group), SMAD4 overexpression plasmid (SMAD4 group) and RUNX3 and SMAD4 overexpression plasmid (RUNX3+SMAD4 group) respectively, using SW480 cells transfected with negative control plasmid as control group. In each group of cells, the changes in RUNX3 and SMAD4 expressions were determined by Western blot and qRT-PCR, and the differences in proliferative and invasion abilities were examined by CCK-8 assay and Transwell assay, respectively.Results The protein expression level of RUNX3 in colorectal cancer tissue was significantly lower than that in adjacent normal tissue, and the mRNA and protein expression levels of SMAD4 were significantly higher in colorectal cancer tissue than those in adjacent normal tissue (all P<0.05); RUNX3 protein expression was negatively correlated with SMAD4 mRNA and protein expressions in colorectal cancer tissue (r=0.511, P=0.004; r=0.487, P=0.009). Compared with control group, the RUNX3 protein expression was significantly increased, while the mRNA and protein expressions of SMAD4 were significantly decreased in the RUNX3 group (all P<0.05); the RUNX3 protein expression showed no significant change (P>0.05), while the mRNA and protein expressions SMAD4 were significantly increased in SMAD4 group (all P<0.05); the RUNX3 protein expression was significantly increased (P<0.05), while the mRNA and protein expressions SMAD4 showed no significant changes in RUNX3+SMAD4 group (both P>0.05). The cell proliferation and invasion abilities were significantly lower in RUNX3 group and were significantly higher in SMAD4 group than those in control group (all P<0.05); both cell proliferation and invasion abilities in RUNX3+SMAD4 represented an intermediate state between RUNX3 group and SMAD4 group, which showed no significant differences with control group (both P>0.05).Conclusion The expression of RUNX3 is down-regulated in colorectal cancer tissue. RUNX3 up-regulation can suppress the malignant biological behavior of colorectal cancer cells, and the mechanism may be associated with its inhibiting the activity of the TGF-β/SMAD4 signaling pathway.