Acquired resistance to gemcitabine (GEM) remains a major barrier in cholangiocarcinoma (CCA) therapy. Melatonin has multi-targeted antitumor activity but is limited by rapid elimination and low oral bioavailability. We developed a maltodextrin-based melatonin-loaded proniosome (MPN) and evaluated its physicochemical properties, ex vivo permeation, and antitumor activity, alone and with GEM, in a GEM-resistant CCA model. MPN was prepared by a provesicular slurry method and characterized for physicochemical properties, stability, molecular dynamics (MD), and ex vivo permeation. Efficacy and tolerability were assessed in mice bearing KKU-213B ^GemR xenografts, and molecular changes by tumor proteomics, mapping onto an independent human intrahepatic CCA single-cell atlas, and computational target analysis. MPN formed spherical vesicles (134.87 ± 2.45 nm; entrapment efficiency 81.39%; loading 44.29%) that retained melatonin content for six months. MD simulations supported hydrophobic interactions with Span 60/cholesterol, and ex vivo flux was approximately 15.7-fold higher than that of free melatonin. GEM + MPN-Low produced the greatest reduction in tumor growth and significantly suppressed Ki-67 expression (p  <  0.0001), without altering serum ALT or creatinine. Proteomics showed lower Sonic Hedgehog abundance in GEM + MPN-treated tumors. Network pharmacology identified 28 candidate targets; docking favored MMP9 (−7.69 kcal/mol) and CDK2 (−7.21 kcal/mol), and 5 × 100-ns simulations indicated a stable complex with MMP9 but a less stable one with CDK2. These predictions were not functionally validated. Overall, MPN enhanced melatonin permeation ex vivo and was associated with an improved response to GEM in resistant CCA xenograft model, supporting further pharmacokinetic and mechanistic studies of oral delivery for biliary tract cancer.