α-Mangostin-loaded mucoadhesive nanoparticles (NPs) were prepared for colon-targeted drug delivery against colorectal cancer cells using pH-dependent composite mucoadhesive NPs. Chitosan (CS) and thiolated chitosan (TCS) were used to form the NPs, following by genipin (GP) crosslinking and the surface modification by Eudragit L100 (L100). The particle size, morphologies and characteristics of NPs were observed. The α-mangostin loading and release patterns were investigated. mucoadhesive properties were examined by the wash-off method. In addition, the anti-tumour activity was tested on colorectal cancer cells. The results showed that NPs were slightly oblong in shape with particle size ranging between 300 and 900 nm. The small size of NPs was found with TCS and larger NPs were observed by GP and L100 process. However, GP and L100 provided an increase in α-mangostin loading, limited the release of α-mangostin in the upper gastrointestinal tract, and enhanced α-mangostin delivery to the colon. The TCS-based NPs with GP and L100 exhibited strong mucoadhesion to colon mucosa, more than uncoated-NPs and CS-based NPs. Moreover, NPs exhibited the anti-tumour activity. Therefore, the mucoadhesive TCS-based NPs could be a promising candidate for a controlled-release drug delivery system of α-mangostin to the colon.