Background: Regorafenib is approved for the treatment of refractory metastatic colorectal cancer (mCRC). However, the molecular mechanisms of inherited or acquired resistance to regorafenib are not well understood. Circulating tumor cells (CTCs) have been shown to be a poor prognostic marker for mCRC patients. We tested whether CTC enumeration and molecular characterization with mRNA levels of EGFR, EpCAM, CEA, and epithelial-mesenchymal transition (EMT) markers will predict outcome and identify molecular escape mechanisms in patients undergoing regorafenib therapy. Methods: A total of 50 patients [male/female; 22/28, median age; 69, median follow-up; 180 days, PR/SD/PD/NE; 1/17/26/6, median progression-free survival (PFS); 69 days, median overall survival (OS); 192 days] with refractory mCRC were enrolled. CTC enumeration was performed at baseline, day21 after initiation of regorafenib, and at the time of treatment intolerance or progression of disease (PD) using the CellSearch System. Poly(A) mRNA was extracted from CTCs. CTC gene expression of EGFR, EpCAM, CEA, and EMT markers (PI3K-α, Akt-2, and TWIST1) was analyzed by a multiplex-PCR based DNA Chip. Results: Patients with < 3 CTCs at baseline and day21 had better outcomes than those with ≥ 3 CTCs (PFS: p = 0.039 and p = 0.032; OS: p < 0.001 and p = 0.007, respectively). Regorafenib treatment significantly increased EGFR expression at PD compared to baseline (p < 0.001). In 64% patients, CTC EGFR gene expression was up-regulated at day 21 (p = 0.004, McNemar's Test) and/or PD (p = 0.011 McNemar's Test). Conclusions: Our study is the first to demonstrate that CTC measurement and characterization may be a useful surrogate for regorafenib efficacy. Furthermore, induced CTC EGFR expression may be a molecular escape mechanism. Therefore, combining regorafenib with anti-EGFR mAbs may have synergistic therapeutic effects.