A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).