Pancreatic lipase (PL) is a key enzyme related to the prevention and treatment of obesity. The aim of the study was to evaluate the inhibitory effects of mulberry leaf polysaccharides (MLP) on PL and possible interaction mechanism, inhibition on lipid accumulation in vitro and in vivo. The results revealed that MLP had obvious inhibitory effects on PL (P < 0.05). The interaction of MLP-PL complexes was in a spontaneous way driven by enthalpy, and hydrogen bonds were the main factors in the binding. MLP could significantly inhibit the development of lipid accumulation in HepG2 cells (P < 0.05). Furthermore, consumption of high-fat diet containing MLP showed protective effects on liver and adipose tissue damages in mice, and inhibited the lipid absorption in digestive tract. MLP also significantly reduced the increased expression level of pancreatic digestive enzymes (P < 0.05). The study indicated that the anti-obesity effect of MLP might be caused by inhibition of lipid absorption via reducing PL activity.