Objective: To investigate the effect of the folate-conjugated nanoparticles (drug carrier) loaded with 5-fluorouracil (5-FU) against bile duct carcinoma and its safety. Methods: The effects of nanoparticles without modification and folate-conjugated nanoparticles or their 5-FU loaded ones on the growth of the cholangiocarcinoma QBC939 cells in vitro were determined by MTT assay. In nude mice bearing QBC939 cell grafts, the inhibitory effects on the tumor grafts among 5-FU, 5-FU loaded nanoparticles and 5-FU loaded folate-conjugated nanoparticles were compared using normal saline as a control. The toxic effects of the folate-conjugated nanoparticles were tested by incubation of the rat liver BRL3A cells and renal NRK cells with different concentrations of the folate-conjugated nanoparticles for 24 h. Results: The in vitro experiments showed that both nanoparticles with or without modification exerted no obvious influence on the growth of QBC939 cells, but both had stronger inhibitory effects than that of 5-FU alone on the growth of QBC939 cells after they were loaded with 5-FU, which was more evident for the folate-conjugated nanoparticles (all P<0.05). The suppression ratios of the tumor grafts in nude mice for 5-FU, 5-FU loaded nanoparticles and 5-FU loaded folate-conjugated nanoparticles were 56.0%, 61.5% and 74.4% respectively, and the differences among them had statistical significance (all P<0.05). There were no morphological changes in BRL3A cells or NRKP cells after exposure to any of the used concentrations of folate-conjugated nanoparticles. Conclusion: The drug delivery system of folate-conjugated nanoparticles has favorable and specific targeting characteristcs, and shows remarkable therapeutic potential for cholangiocarcinoma after it is loaded with 5-FU.