In this work, lipase-catalyzed acidolysis of microbial oil (MO) from Mortierella alpina with caprylic acid (CA) was carried out in a solvent-free system to produce structured lipids (SLs) enriched with medium-chain fatty acids (MCFAs). Among four commercial immobilized lipases screened for their acidolysis efficiency, Lipozyme RM IM from Rhizomucor miehei displayed the highest ratio of CA incorporation. The in fl uence of enzyme loading, substrate molar ratio, reaction temperature, and reaction time on the CA incorporation was investigated. The produced SLs contained 49.45% arachidonic acid at the sn-2 position and 29.7% CA at sn-1,3 positions. In addition, a total of 25 triacylglycerol (TAG) molecular species containing CA were generated following the enzymatic acidolysis by using ultra-performance liquid chromatography with quadrupole-time-of- flight mass spectrometry (UPLCQ-TOF-MS). The most abundant TAG species in the SLs were (8:0-20:4-8:0), (20:4-20:4-8:0), and (20:3-20:4-8:0) accounting for 11.23%, 9.42%, and 4.99%, respectively. Moreover, the SLs showed an obvious decrease in the melting and crystallization pro fi les in comparison with those of the native MO. The synthesized SLs containing MCFAs would have the combined advantages of providing quick energy and as a source of essential fatty acids.