Abstract: Candida rugosa contains several lipase (CRLs) genes, and CRLs show diverse enzyme activity despite being highly homologous across their entire protein family. Previous studies found that LIP4 has a high esterase activity and a low lipolytic activity and lacks interfacial activation. To investigate whether the C-terminal region of the CRLs mediates enzymatic activity, chimeras were generated in which the C-terminus of LIP4 from either residue 374, 396, 417, or 444 to residue 534 was swapped with the corresponding peptide from the isoform LIP1. A chimeric lipase containing the C-terminus from 396 to 534 of LIP1 on a LIP4 scaffold showed activity similar to that of commercial CRL on triolein, and lipolytic activity increased 2–6-fold over that of LIP4. Moreover, interfacial activation was also observed in the chimeric lipase. To improve its enzymatic properties, a novel glycosylation site was added at residue 314. The new glycosylated lipase showed improved thermostability and enhancement in enzymatic activity, indicating its potential for use in further application.