Hydrogen sulfide (HS) is a novel adipokine mediating glucose uptake, lipid storage and mobilization, thus contributing to the genesis of obesity and associated diseases. Our previous work demonstrated that HS inhibited isoproterenol-stimulated lipolysis by reducing the phosphorylation of perilipin 1 (plin-1), a lipid-droplet protein blocking lipase access. How HS modulates plin-1 phosphorylation is still unclear. Our present study found that an HS donor slightly increased adipose tissue weight and reduced lipolysis in mice; by contrast, deleting the key HS generation enzyme cystathionine gamma lyase (CSE) in adipocytes lowered adipose accumulation and enhanced lipolysis. Intriguingly, an HS donor induced sulfhydration of plin-1 but not hormone-sensitive lipase, and CSE deletion abolished the post-translational modification of plin-1. During isoproterenol-stimulated lipolysis, plin-1 sulfhydration was associated with reduced phosphorylation, and removing sulfhydration by dithiothreitol recovered the phosphorylation. Finally, plin-1 knockout abolished the effect of HS on lipolysis, which indicates that plin-1 sulfhydration is a major direct target of HS in lipolysis. We have identified a new post-translation modification, sulfhydration (direct action by HS) of plin-1, causing reduced phosphorylation then decreased lipolysis. This finding also highlights a novel molecular regulatory mechanism of lipolysis.