Nanoparticles (NPs) modified with glycocholic acid (GCA) at surfaces are considered promising tools to overcome oral drug delivery barriers. However, question on the influence of surface GCA density over delivery efficiency arises due to ligand-induced changes in surface properties of NPs. To answer this question, we engineered GCA-modified nanoparticles (GCA NPs) with different surface densities, and their surface hydrophobicity, mucus penetration, cellular uptake, apparent permeability coefficient (P), intracellular trafficking behavior and oral bioavailability were compared. The results turned out that GCA NPs were found with increased surface hydrophobicity, which limited their transport across mucus layer. Nanoparticles with 50 % surface GCA density (50 % GCA NPs) demonstrated optimal performance. Compared to 100 %GCA NPs, 50 %GCA NPs exhibited an 83.28 % improvement in mucosal penetration capability, while a 250.67 % increase in cellular uptake was noticed when comparing with PEG NPs. Overall, the 50 % GCA NPs significantly enhanced trans-epithelial transport across Caco-2/E-12 co-cultured monolayers, achieving a Pvalue of 3.30 × 10 cm/s, while the Pvalues of 100 %GCA NPs and PEG NPs were 2.23 × 10 cm/s and 1.73 × 10 cm/s. In vivo studies confirmed their therapeutic potential: oral administration of semaglutide (SMG)-loaded 50 % GCA NPs increased systemic SMG bioavailability to 12.12 %, achieving sustained glycemic control in type 2 diabetic rats. The optimized formulation exhibited favorable safety profiles and prolonged pharmacological effects. This study establishes a critical equilibrium between enhanced mucosal penetration and ligand-receptor interaction, while mitigating intracellular retention issues associated with high-density GCA-functionalized nanoparticles. These findings indicate that attentions should be paid in ligand-associated surface property alterations, which could exert unexpected influence on the performance of NPs constructed for oral peptide drug delivery.