Semaglutide, a glucagon-like peptide-1 (GLP-1) analog, extends its half-life by reversibly binding to human serum albumin (HSA). To investigate binding interactions at various fatty acid sites, we performed 1.5 μs conventional molecular dynamics (cMD) simulations and molecular mechanics generalized Born surface area (MM-GBSA) free energy calculations. MM-GBSA results show that the FA3-FA4 site (referred to as FA3) is the most favorable binding site (= －75.33 ± 15.04 kcal/mol), followed by the FA1 and FA6 sites, consistent with findings from previous two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. At the FA3 site, electrostatic interactions predominated, forming the most hydrogen bonds and leading to a more compact structure, with no significant changes in residue-residue contacts compared to HSA without semaglutide. Next, Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations were performed to enhance semaglutide's flexibility by selectively increasing its potential energy. Key residues, R348 and R485, were identified as crucial for localizing the C18 dicarboxylic acid side chain. These findings provide valuable insights into HSA-Semaglutide binding, potentially guiding the rational design of lipidated albumin-binding peptides in future studies.