Abstract: To investigate the molecular characteristics and functional mechanism of glutamine synthetase (GS), a key rate-limiting enzyme in plant nitrogen assimilation, in pecan (Carya illinoinensis), the cytoplasmic CiGS1.1 gene was cloned. Its full-length cDNA is 1071 bp and encodes 356 amino acids The results of bioinformatics, phylogenetic, and quantitative real-time PCR analyses showed that CiGS1.1 protein exhibits a molecular weight of 39.21 kDa, a theoretical isoelectric point of 5.79, strong hydrophilicity and structural stability, with no transmembrane domains detected. Secondary structure prediction indicated a composition of 28.65% α-helix, 17.98% extended strand, and 53.37% random coil. CiGS1.1 shares the closest evolutionary relationship with JrGS1.1/1.2 from English walnut (Juglans regia), and notably lacks the C-terminal regulatory motif characteristic of Arabidopsis thaliana homologs. Subcellular localization confirmed its cytoplasmic distribution. CiGS1.1 showed the highest expression level in fruits, which was significantly higher than that in leaves and roots (P < 0.05).. Thus, this study characterized the molecular properties and tissue expression pattern of CiGS1.1.