Background: Intracellular calcium (Ca2+) homeostasis plays an essential role in adipocyte metabolism and its alteration is associated with obesity and related disorders. Transient receptor potential vanilloid 4 (TRPV4) channels are an important Ca2+ pathway in adipocytes and their activity is regulated by metabolic mediators such as insulin. In this study, we evaluated the role of TRPV4 channels in metabolic activity and adipokine secretion in human white adipocytes. Methods: Human white adipocytes were freshly cultured and the effects of the activation and inhibition of TRPV4 channels on lipolysis, glucose uptake, lactate production, and leptin and adiponectin secretion were evaluated. Results: Under basal and isoproterenol-stimulated conditions, TRPV4 activation by GSK1016709A decreased lipolysis whereas HC067047, an antagonist, increased lipolysis. The activation of TRPV4 resulted in increased glucose uptake and lactate production under both basal conditions and insulin-stimulated conditions; in contrast HC067047 decreased both parameters. Leptin production was increased, and adiponectin production was diminished by TRPV4 activation and its inhibition had the opposite effect. Conclusion Our results suggested that TRPV4 channels are metabolic mediators involved in proadipogenic processes and glucose metabolism in adipocyte biology. TRPV4 channels could be a potential pharmacological target to treat metabolic disorders.