Background and PurposeDuring angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre‐existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3‐mercaptopyruvate sulfurtransferase (3‐MST), an important H2S‐producing enzyme in ECs. The aim of our study was to explore the potential role of 3‐MST in human EC bioenergetics, metabolism, and angiogenesis.Experimental ApproachTo assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA‐mediated 3‐MST attenuation and pharmacological inhibition of proliferation, migration, and tube‐like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds.Key Results3‐MST‐attenuated ECs subjected to shRNA or pharmacological inhibition of 3‐MST significantly reduced EC proliferation, migration, and tube‐like network formation. 3‐MST silencing also suppressed VEGF‐induced EC migration. From bioenergetic and metabolic standpoints, 3‐MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome.Conclusion and Implications3‐MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3‐MST pathway may be a potential candidate for therapeutic modulation of angiogenesis.Linked ArticlesThis article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc