AbstractMany population studies have shown that blood concentrations of high‐density lipoprotein (HDL) cholesterol are inversely correlated with risk of cardiovascular disease (CVD). However, in recent studies, increasing blood HDL cholesterol concentrations failed to reduce CVD events. On the other hand, studies suggest that improving HDL quality can be a more efficient tool for assessing atherosclerotic risk than simply measuring blood HDL cholesterol concentration. Thus, improving HDL activity using natural substances might be a useful therapeutic approach to reducing CVD risk. We previously isolated a novel active compound from Nannochloropsis microalgae termed lyso‐diacylglyceryltrimethylhomoserine (lyso‐DGTS), which increased activity of paraoxonase 1, the main antioxidant enzyme associated with HDL. Here we examined the effect of lyso‐DGTS on HDL quality and function. Tryptophan‐fluorescence‐quenching assay showed that lyso‐DGTS interacts spontaneously with the entire HDL lipoprotein and with apolipoprotein A1 (ApoA1), the major structural and functional HDL protein, with high affinity (Ka = 2.17 × 104 M−1 at 37°C). Lyso‐DGTS added to HDL and to ApoA1 increased cholesterol efflux from macrophage cells, the main antiatherogenic function of HDL, dose‐dependently, and significantly increased HDL's ability to induce nitric oxide production from endothelial cells. In‐vivo supplementation of lyso‐DGTS to the circulation of mice fed a high‐fat diet via osmotic mini‐pumps implanted subcutaneously enhanced HDL anti‐inflammatory effect significantly as compared to controls. Our findings suggest that lyso‐DGTS may have a beneficial effect in decreasing atherosclerosis risk by interacting with HDL particles and improving their quality and antiatherogenic functions.