Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca²⁺ ([Ca²⁺]_i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca²⁺-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca²⁺]_i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca²⁺ imaging. We found that histamine increased [Ca²⁺]_i was maintained at a static elevated level after a transient peak. The elevated Ca²⁺ plateau was vanished when extracellular Ca²⁺ was removed, indicating Ca²⁺ influx from extracellular mediated the histamine-induced Ca²⁺ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca²⁺ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca²⁺ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.