Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target.