Subsequent to a rise in intracellular Ca2+ ([Ca2+]i), hyperpolarization of the endothelium coordinates vascular smooth muscle relaxation along resistance arteries during blood flow control. In the lymphatic vasculature, collecting vessels generate rapid contractions coordinated along lymphangions to propel lymph, but the underlying signalling pathways are unknown. We tested the hypothesis that lymphatic endothelial cells (LECs) exhibit Ca2+ and electrical signalling properties that facilitate lymph propulsion. To study electrical and intracellular Ca2+ signalling dynamics in lymphatic endothelium, we excised collecting lymphatic vessels from the popliteal fossa of mice and removed their muscle cells to isolate intact LEC tubes (LECTs). Intracellular recording revealed a resting membrane potential of ∼−70 mV. Acetylcholine (ACh) increased [Ca2+]i with a time course similar to that observed in endothelium of resistance arteries (i.e. rapid initial peak with a sustained “plateau”). In striking contrast to the endothelium‐derived hyperpolarization (EDH) characteristic of arteries, LECs depolarized (>15 mV) to either ACh or TRPV4 channel activation. This depolarization was facilitated by the absence of Ca2+‐activated K+ channels (KCa) as confirmed with PCR, persisted in the absence of extracellular Ca2+, was abolished by LaCl3 and was attenuated ∼70% in LECTs from Trpv4−/− mice. Computational modelling of ion fluxes in LECs indicated that omitting K+ channels supports our experimental results. These findings reveal novel signalling events in LECs, which are devoid of the KCa activity abundant in arterial endothelium. Absence of EDH with effective depolarization of LECs may promote the rapid conduction of contraction waves along lymphatic muscle during lymph propulsion. This article is protected by copyright. All rights reserved