FadL is an important member of the family of fatty acid transport proteins within membranes. In this study, I I conventional molecular dynamics (CMD) and 25 steered molecular dynamics (SMD) simulations were performed to investigate the dynamic mechanism of transport of long-chain fatty acids (LCFAs) across FadL. The CMD simulations addressed the intrinsically dynamic behavior of FadL. Both the CMD and SMD simulations revealed that a fatty acid molecule can move diffusively to a high-affinity site (HAS) from a low-affinity site (LAS). During this process, the swing motion of the L3 segment and the hydrophobic interaction between the fatty acid and FadL could play important roles. Furthermore, 22 of the SMD simulations revealed that fatty acids can pass through the gap between the hatch domain and the transmembrane domain (TMD) by different pathways. SMD simulations identified nine possible pathways for dodecanoic acid (DA) threading the barrel of FadL. The binding free energy profiles between DA and FadL along the MD trajectories indicate that all of the possible pathways are energetically favorable for the transport of fatty acids; however, one pathway (path VI) might be the most probable pathway for DA transport. The reasonability, and reliability of this study were further demonstrated by correlating the MD simulation results with the available mutagenesis results. On the basis of the simulations, a mechanism for the full-length transport process of DA from the extracellular side to the periplasmic space mediated by FadL is proposed.