Mucosal drug delivery plays an increasing role in the clinical setting owing to mucin's advantageous biochemical and pharmacological properties. However, how this transport system recognizes different substrates remains unclear. In this study, we explore the mechanism of bioactive (quercetin and berberine) promiscuity of mucin using various spectroscopic techniques and molecular dynamics simulations. The UV−visible spectroscopy results and the decreased fluorescence intensity of mucin in the presence of the bioactive compounds via a static quenching mechanism confirmed ground-state complex formation between the bioactives and mucin. The binding constants (K b ) were evaluated at different temperatures to afford K b values of ∼10 4 Lmol −1 , demonstrating the moderate and reasonable affinity of the bioactives for mucin, yielding greater diffusion into the tissues. Thermodynamic analysis and molecular dynamics (MD) simulations demonstrate that mucin−bioactive complex formation occurs primarily because of electrostatic/ionic interactions, while hydrophobic interactions were also crucial in stabilizing the complex. Far-UV circular dichroism spectroscopy showed that bioactive binding induced secondary structural changes in mucin. Sitemap and MD simulation indicated the principal binding site of mucin for the bioactives. This study also provides insight into the bioactives promiscuity of mucin in the presence of a crowded environment, which is relevant to the biological activity of mucin in vivo. An in vitro drug release study revealed that crowding assisted drug release in an enhanced burst manner compared with that in a dilute buffer system. This work thus provides fresh insight into drug absorption and distribution in the native cellular environment and helps direct new drug design and use in pharmaceutical and pharmacological fields.