To obtain mulberry leaf protein peptides with high lipid-lowering activity, this study used mulberry leaf crude proteins-Da10 glutelin (DSG), Da10 albumin (DS), and Tang10 albumin (TS)-as well as ultrafiltered albumin (UDS) and glutelin (USG), as substrates. These proteins were hydrolyzed with a neutral protease to produce the hydrolysates NDSG, NDS, NTS, NUDS, and NUSG. The inhibitory capacities of NDSG and ADSG (i.e., the enzymatic hydrolysates obtained by hydrolyzing DSG with neutral protease and alkaline protease, respectively) on pancreatic lipase (PL) and cholesterol esterase (CE) were compared. Single-factor experiments combined with orthogonal optimization were conducted to determine the optimal extraction process for mulberry leaf lipid-lowering peptides, which yielded the optimized product HDG. A non-alcoholic fatty liver disease (NAFLD) steatosis cell model was established to investigate the intracellular lipid-lowering effects of HDG through Oil Red O staining and measurements of intracellular total cholesterol (TC) and triglyceride (TG) levels. Results showed that the PL inhibition rate of NDSG was significantly higher than that of other groups (P<0.05), and also higher than that of its ultrafiltration group NUDS. The CE inhibition rate of NDSG was significantly higher than that of ADSG; hence, neutral protease was selected for optimizing the enzymatic hydrolysis process. Orthogonal optimization revealed that the optimal preparation conditions for lipid-lowering peptides from mulberry leaf protein were as follows: enzymatic hydrolysis with a neutral protease for 2 h, an enzyme-to-substrate ratio of 4%, a substrate concentration of 1%, and a pH of 7.5. Under these conditions, the enzymatic hydrolysate HDG exhibited a PL inhibition rate of 85.68% and a CE inhibition rate of 69.49%. Moreover, HDG significantly inhibited lipid droplet accumulation in HepG2 cells and reduced intracellular TG and TC levels (P<0.05), thereby alleviating cellular steatosis. This study successfully optimized the preparation process for mulberry leaf glutelin peptides with notable lipid-lowering activity, thus laying a foundation for further research and industrial production of mulberry leaf protein-derived lipid-lowering peptides.