A novel lipid inhibition peptide Leu-Leu-Val-Val-Try-Pro-Trp-Thr-Gln-Arg (PP1) (MW 1274.53 Da) was obtained from Chlorella pyenoidose using enzymatic hydrolysis, gel filtration chromatography, and LC-MS/MS. Its lipid inhibition effects indicated that the synthetic peptide PP1 exhibits a good inhibitory effect against porcine pancreatic lipase (PL) (47.95%) at 200 μg/mL, which could be attributed to its hydrogen binding into catalytic sites of PL (Ser153, Asp177, and His 264) by docking analysis. Furthermore, in 3T3-L1 cells, the synthetic PP1 remarkedly decreased the accumulation of intracellular triacylglycerol (27.9%, 600 μg/mL), which carried a similar consequence as the positive drug simvastatin (24.1%, 10 μM). Western blot revealed that PP1 inhibited the lipid accumulation and fatty acid synthesis in 3T3-L1 adipocytes in two pathways, primarily: nonalcoholic fatty liver disease (NAFLD) pathway (C/EBPα, SREBP-1c, AMPKα) and AMPK signaling pathway (SREBP-1c, PPARγ, AMPKα). In short, these results support that PP1 can be used as a potential agent against obesity.