Lipolysis occurs during ripening of dairy products as a result of esterase/lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria in comparison to other species. However, in cheeses with extended ripening periods, lipolytic LAB may have potential advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but without previous reports on the production of lipases. The aim of this project was purified, characterize and identify the esterase produced by P. acidilactici isolated from Gouda cheese and determine its phospholipid hydrolysis profile, with a focus on increased absorption of these in the human gut. To purify the lipolytic protein, zymography was performed in native conditions. The band that displayed activity was excised from the gel, concentrated by ultrafiltration and sent for sequence analysis by LC-MS/MS. Also, this fraction was subject to biochemical characterization as a function of: pH, temperature, ions, hydrolysis of different substrates, and activity over phospholipids. A single protein with lipolytic activity was detected by zymography, with a molecular weight of 86 kDa. The peptides found by LC-MS/MS indicate it is a putative metallophosphoesterase with a theoretical molecular weight of 45.5-kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity from pH 8.0 to 9.0. The optimal temperature for activity was 37°C and it lost 15% of activity after incubation at 90°C for 1 h. This lipase showed activity over short chain fatty acids and exhibited high hydrolysis of PG (phosphotidylglycerol) and PI (phosphotidylinositol). It also hydrolyzed PS, PC and PE (phosphotidylserine, phosphotidylcholine, and phosphotidylethanolamine, respectively) but with less effect. This is the first lipase reported for Pediococcus genus isolated from a dairy product and shown to hydrolyze phospholipids, which may exert human health benefits through increased digestibility in intestinal cells