A series of twenty-three parthenolide-5-fluorouracil (5-FU) conjugates ware synthesized and evaluated for their anti-cancer activities against human hepatocellular carcinoma cell line Bel-7402 and 5-fluorouracil resistant human hepatocellular carcinoma cell line Bel-7402/5-FU. The preliminary structure-activity relationships were discussed. The most active compound 15d showed high activity against Bel-7402/5-FU cell line with IC50 value of 2.25 μM, which demonstrated 5.8-fold improvement compared to that of the parent compound parthenolide (IC50 = 12.98 μM). The investigation of preliminary molecular mechanism of 15d revealed that 15d could reverse drug resistance by inhibiting MDR1, ABCC1 and ABCG2 to increase the intracellular drug accumulation and induce apoptosis of Bel-7402/5-FU cells through mitochondria mediated pathway. On the base of these results, compound 15d is deserved to be further investigated as a potential anti-HCC lead compound for ultimate discovery of pathenolide-based anti-cancer drug.