Diabetic patients have lower blood levels of L-cysteine (LC) and hydrogen sulfide (H2S) and a higher incidence of vascular inflammation. This study examined whether impaired LC or H2S levels affect vascular inflammation markers in diabetes. Human U937 monocytic cells were treated with high-glucose (HG, 25 mM, 20 h) in the presence or absence of LC (100, 500, or 1,000 µM, an endogenous precursor of H2S) or Na2S (5 or 25 µM, an exogenous source of H2S). Both LC and Na2S supplementation decreased intracellular ROS production and increased cellular PIP3 (phosphatidylinositol-3,4,5-trisphosphate) in HG-exposed cells. The effect of LC on PIP3 was prevented by propargylglycine, an inhibitor of cystathionine-γ-lyase (CSE) that catalyzes H2S formation from LC. Signal silencing studies with CSE siRNA also showed the inhibition of H2S formation and PIP3 upregulation in LC-supplemented CSE knockdown cells exposed to HG. This demonstrates that H2S plays a role in mediating the effect of LC on increased PIP3. Using the PI3K specific inhibitor LY294002, this study demonstrated that PI3K activation mediates the effect of LC and Na2S on PIP3 upregulation. Results showed that supplementation with LC and Na2S reduced NF-κB phosphorylation and the secretion of TNF-α, MCP-1, IL-8, IL-1β, and IP-10. Treatment with LC (500 µM), Na2S (25 µM), and PIP3 (5 nM) increased the AMPK phosphorylation and PPARγ expression in cells exposed to HG. This study reports for the first time a novel molecular mechanism by which Na2S or LC supplementation can lower oxidative stress and various markers of vascular inflammation in diabetes. J. Cell. Biochem. 114: 2334–2345, 2013. © 2013 Wiley Periodicals, Inc.