Sphingolipids (SLs) are vital for cells as forming membrane and transducing signals. The first step for de novo biosynthesis of SLs is catalyzed by the pyridoxal-5’-phosphate (PLP)-dependent enzyme Serine palmitoyltransferase (SPT), which has been proven to be a promising drug target for treating various diseases. However, there are few SPT-specific inhibitors have been identified so far. Myriocin, a natural fungal product, is confirmed as the most potent inhibitor of SPT and has been widely used, but studies of its molecular mechanism are still underway. Besides, there is no intact co-crystal structure of SPT binding Myriocin until now. Aiming to uncover the interaction mechanism between SPT and PLP binding Myriocin at the molecular level, a systematic computational strategy was performed in this present study. Firstly, covalent docking was implemented to preliminarily predict the binding pose SPT/PLP-Myriocin aldimine and its structurally similar intermediate SPT/PLP-β-ketoacid aldimine. Secondly, two binding complexes were treated as initial structures to perform molecular dynamics simulations and binding free energy calculations. The calculated docking scores and predicted binding energies were consistent with the reported bioactivities. Finally, the binding mechanism of Myriocin binding with SPT was meticulously described and the key residues making favorable contributions were highlighted. Taken together, the current study could provide some important information and valuable guidance for further rational screening, design, and modification of potent specific SPT inhibitors.