Claudins (CLDNs) are tight junction proteins frequently displaying abnormal expression patterns in cancer. Overexpression of CLDN2, a frequent event in colorectal cancer, increases tumourigenicity and hampers differentiation. Here, we first show that overexpression of claudin-2 in the SW480 colorectal cancer cell line, which expresses negligible endogenous levels of this protein, strongly increased their self-renewal in vitro. Conversely, shRNA-mediated CLDN2 depletion inhibited the tumour-initiating potential of LGR5-positive DLD1 cells without affecting that of LGR5-negative cells. Increased staining for CK20 following CLDN2 down-regulation was detected in a colonosphere model, suggesting that these cells are engaged towards differentiation. Expression of mRNAs encoding ALDH1A1, BMI1, LGR5, CD44, ErbB receptor subtypes and proteins involved in Epithelial-Mesenchyme Transition was strongly down-regulated following CLDN2 down-regulation in DLD1 and T84 cell lines. We also analyzed the impact of claudin-2 depletion on the phenotype of cells displaying high aldehyde dehydrogenase (ALDH) activity. The percentage of ALDHhigh cells was significantly reduced following transfection of DLD1 and T84 cells with CLDN2-selective siRNA, suggesting that CLDN2 may regulate the tumour-initiating potential of colorectal cancer cells by controlling the equilibrium between ALDHhigh and ALDHlow populations. To validate this hypothesis, we used flow cytometry to sort ALDHhigh and ALDHlow cells from the T84 cell line, transfected them with siRNA directed against b-galactosidase or CLDN2, and grew these cells under 2D conditions in the presence of serum for 72h before re-analyzing ALDH activity in these cells. Under these conditions, homogenous ALDHHigh and low populations both generate heterogeneous populations, and CLDN2 depletion slows down the rate of ALDHlow to high conversion and accelerates that of ALDHhigh to low conversion, suggesting that CLDN2 is an important factor in the maintenance of phenotypic equilibrium at population level in colorectal cancer cells. Our results suggest that CLDN2 overexpression in colorectal cancer promotes the tumour-initiating cell phenotype at least in part regulating phenotypic transitions between TICs and non-TICs.