<p>Nanostructured lipid carriers (NLCs) decorated with sulfhydryl-modified surfactants have recently gained attention for delivering BCS Class IV drugs. However, the impact of the chain-length of these surfactants on the permeation and bioavailability properties of NLCs is still unknown. Therefore, this study investigates the effect of surfactant chain-length on the mucoadhesive, permeation, and bioavailability properties of NLCs. For this purpose, short- and long-chain sulfhydryl-modified polyethoxylated surfactants were generated to develop mucoadhesive NLCs and loaded with the model drug aprepitant (APT). NLCs were characterized and assessed for comprehensive physicochemical and biological evaluations. Moreover, in-vivo studies were performed for proof-of-concept to show enhanced oral drug bioavailability. NLCs showed particle size under 200&#xa0;nm with 6.9 and 6.7% drug loading and 85 and 84% drug entrapment for short- and long-chain surfactants, respectively. The drug-loaded NLCs were safe and stable, and short- and long-chain surfactants containing NLCs exhibited 11.6- and 9.6-fold enhanced mucoadhesion, respectively. Moreover, in comparison to long-chain sulfhydryl-modified surfactant, short-chain surfactant is transported into deeper segments of mucus due to less interaction with the mucus. Similarly, short-chain sulfhydryl-modified surfactants showed significantly enhanced cellular permeation across Caco-2 cell lines. Furthermore, the long-chain sulfhydryl-modified surfactants showed 4.38-fold enhanced C_max, whereas due to better diffusion and mucoadhesion properties, the short-chain surfactants exhibited 5.38-fold enhanced C_max. Similarly, 34.8% relative bioavailability was attained for short-chain surfactants and 24.8% for long-chain surfactants. These results suggest short-chain sulfhydryl surfactants are promising candidates for improving the oral delivery of poorly soluble drugs and warrant further investigation for clinical translation.</p> Graphical Abstract <p></p>