Background: In the identification of biomarkers for anticancer drugs, the lack of objectivity in the selection of candidate factors makes it difficult to interpret the mechanism. We performed preclinical analysis and translational validation study to identify the candidate cytokines for regorafenib efficacy in metastatic colorectal cancer (mCRC) patients. Methods: As a first preclinical process, we selected candidate cytokines according to our gene chip analysis using a panel of human cancer cell lines (JFCR39). We then validated predictive value of the cytokines in mCRC patients receiving regorafenib (discovery, N=54) and FTD/TPI (control, N=16). Blood samples were obtained at baseline (BL), before second cycle (2nd) and progressive disease (PD), and cytokine levels were measured using ELISA. Finally, we measured changes in cytokine levels and cell number in colorectal cancer cell lines treated with regorafenib. Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier curves. Results: Our gene chip analysis showed association between high matrix metalloproteinase (MMP)-14 expression and high sensitivity to regorafenib, and the MMPs pathway was examined in blood samples. In the discovery cohort, high MMP14 levels at BL (mean 2.66 vs. 1.91 ng/ml, P=0.006) and PD (mean 2.34 vs. 1.71 ng/ml, P=0.02) were associated with tumor shrinkage, and high MMP14 levels at PD were associated with longer PFS (3.7 vs. 2.5 mos, HR0.45, 95%CI: 0.21-0.95, P=0.03). Patients with decreased MMP9 levels at 2nd had higher disease control rate (74% vs. 29%, P=0.002) and tumor shrinkage (52% vs. 13%, P=0.006), longer PFS (4.5 vs. 2.0 mos, HR0.34, 95%CI: 0.18-0.65, P<0.001) and OS (13.6 vs. 5.2 mos, HR0.35, 95%CI:0.18-0.68, P=0.001) than those with increased changes. These findings were not observed in the control cohort, but MMP9 levels decreased at 2nd in 93.8% of patients. In a regorafenib-sensitive cell line, HT29, MMP14 levels were increased whereas there was no change in regorafenib-resistant HCT15 cells. Conclusions: Our preclinical data-based translational validation study suggests that MMP14 and MMP9 may serve as a prognostic marker of regorafenib, and the results also provide insight for novel combination therapy with anti-MMP9 agent or FTD/TPI.