Cisplatin is an anticancer agent and induces DNA interstrand cross-links (ICLs). ICLs activate various signaling processes and induce DNA repair pathways, including the Fanconi anemia (FA) pathway. FA complementation group D2 (FANCD2) is monoubiquitinated in response to DNA damage, leading to activation of the DNA double-strand-break repair protein, RAD51. Caffeine increases the anticancer activity of cisplatin by inhibiting DNA repair; however, details of the mechanism remain unclear. We investigated the mechanism responsible for the synergistic anticancer effect of cisplatin and caffeine in HepG2 human hepatocellular carcinoma cells, focusing on the FA pathway. Caffeine (>= 100 mu g/mL) significantly enhanced the antiproliferative activity induced by 3.8 mu g/mL cisplatin. Caffeine (200 mu g/mL) promoted apoptosis and inhibited the increase in the proportion of viable cells in S phase that occurred in the presence of 3.8 mu g/mL cisplatin. Both FANCD2 monoubiquitination and RAD51 expression were significantly inhibited by co-treatment with 200 mu g/mL caffeine and 3.8 mu g/mL cisplatin compared with cisplatin alone. In conclusion, caffeine enhances the anticancer effect of cisplatin by inhibiting FANCD2 monoubiquitination. In HepG2 cells, caffeine might inhibit the FA pathway and thereby regulate DNA damage responses such as DNA repair and apoptosis.