The recently identified class of microRNAs (miRNAs) provided a new insight in cancer research. As the member of miRNAs family, miR-34a, miR-155 and miR-200c abnormalities have been found in various types of cancer. However, the relationship between these three miRNAs (miR-34a, miR-155 and miR-200c) and colorectal cancer is unclear. In this study, we applied stem-loop real-time PCR to quantitatively detect miR-34a, miR-155 and miR-200c expression in 109 pair-matched human colorectal cancers and the corresponding normal mucosa. MiR-34a (2.2-fold), miR-155 (2.3-fold) and miR-200c (3.1-fold) were all expressed at higher levels in colorectal cancer (P = 0.001, 0.005 and 0.001, respectively). In rectum, miR-34a and miR-200c were significantly upregulated (P = 0.006 and 0.007), while the miR-155 overexpression was not statistically significant (P = 0.083). In colon, the higher expression of three miRNAs was seen, however, without significant difference (P > 0.05). We also found that the miR-34a expression was higher in rectal cancer having more advanced TNM stage (III + IV, P = 0.03). Then miR-200c expression was positively correlated with and sera CEA level of rectal cancer patients (P = 0.04). In conclusion, our results thus suggest that the overexpression of miR-34a, miR-155 and miR-200c be associated with the development of colorectal cancer, meanwhile miR-34a may be involved in the development and progression of rectal cancer. The more deeply and larger scale research are required to prove the correlation.