The expression of the gap junction molecule connexin-45 (Cx45; GJC1) in lymphatic endothelium and its functional relevance were not previously known. We found that Cx45 was expressed widely in the endothelium of murine lymphatics, in both valve and non-valve regions. Cell-specific deletion of Cx45, driven by a constitutive Cre line (Lyve1-Cre) or an inducible Cre line (Prox1-CreERT2), compromised the function of lymphatic valves, as assessed by physiological tests (back leak and closure) of isolated, single-valve vessel segments. The defects were comparable to those previously reported for loss of Cx43 and, like Cx43, deletion of Cx45 resulted in shortening and/or increased asymmetry of lymphatic valve leaflets, providing an explanation for the compromised valve function. In contrast to Cx43, LEC-specific deletion of Cx45 did not alter the number of valves in mesenteric or dermal lymphatic networks, or the expression patterns of the canonical valve-associated proteins PROX1, ITGA9 or CLAUDIN5. Constitutive deletion of Cx45 from LECs resulted in increased backflow of injected tracer in popliteal networks in vivo and compromised the integrity of the LEC permeability barrier in a subset of collecting vessels. These findings provide evidence for an unexpected role of Cx45 in the development and maintenance of lymphatic valves.