AbstractThe lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex‐dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex‐dependent differences therein, in Prox1‐CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1‐CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1‐CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1‐CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1‐CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1‐CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC‐specific role for Panx1 in the functions of the lymphatic system.