Matrix metalloproteinases (MMPs), a family of endopeptidases also known as gelatinases, have been reported to affect the acquisition of the cell proliferative, cell invasive and metastatic phenotype of several types of cancer. In particular, the gelatinases MMP-2 and -9 have been revealed to facilitate tumor growth and invasion in patients with colorectal cancer (CRC). However, it is not known whether the gelatinase activity of MMP-2 and-9 is also elevated in Vietnamese patients with CRC. The activity of MMP-2 and-9 in the tissue samples of 103 patients with CRC was evaluated by gelatin zymography and quantified using ImageJ. The association between the level of activity of MMP-2 and-9 and various clinicopathological factors was analyzed, and Chisio BioPAX Editor software was used to visualize the biological pathways regulating the activity of the MMPs. The present study noticed significantly increased activity of active MMP-2 and MMP-9 in tumor tissues (P < 0.01), and significantly decreased levels of pro-form MMP-2 and MMP-9 in tumor tissues (P < 0.01), compared with that in adjacent tissues in patients with CRC. A correlation between the normalized different activity of MMP-2 and-9 and various clinicopathological features was observed. Furthermore, bioinformatics analysis indicated that the alteration in the activity of MMP-2 and MMP-9 may have been controlled by biological pathways involving the tissue inhibitors of metalloprotease-2 and -1. These findings indicate that the activity of the gelatinases MMP-2 and -9 affects the tumor progression and metastasis of patients with CRC, providing a potential novel approach for determining the prognosis of CRC.