Synthesis of biologically active peptides, alkaloids, and immunosuppresants such as FK506 requires enantiomerically-pure pipecolic acid (2-piperidinecarboxylic acid). We report an efficient kinetic resolution of pipecolic acid esters by enzyme-catalyzed hydrolysis. We screened commercially available hydrolases and identified crude lipase from Aspergillus niger (ANL) as the most enantioselective catalyst for the hydrolysis of (±)-methyl pipecolate, E = 20 ± 4 in favor of the (S)-enantiomer at pH7. Changing of the ester group to n-pentyl or n-octyl did not increase the enantioselectivity, while addition of an N-acetyl group decreased the enantioselectivity. Partial purification of ANL by fractional precipitation with ammonium sulfate (25-45 % saturation) increased the enantioselectivity to >100. A synthetic scale resolution of (±)-n-octyl pipecolate using this partially purified ANL gave (S)-(–)-pipecolic acid (93 % ee, 0.89 g) and (R)-(+)-pipecolic acid (97 % ee,1.20g). Further purification of ANL confirmed that the lipase (apparent molecular weight of 32 kDa), and not an impurity, was responsible for the enantioselective hydrolysis of octyl pipecolate.