The global effects of 5-fluorouracil (FU) on cervical carcinoma cells were analyzed using an efficient proteomic method. More than 50 proteins showed a significant change in 5-FU-treated cervical carcinoma cells compared to control cells. Among them, 34 proteins have been identified by employing two-dimensional gel electrophoresis and MALDI-TOF-MS using peptide mass fingerprinting. In results, 22 proteins were upregulated (CIDE-B [cell death-inducing DFFA-like effector B], caspase-3, caspase-8, Apo-1/CD95 (Fas), etc.) and 12 proteins were downregulated (mitotic checkpoint protein BUB3, myc proto-oncogene protein [c-myc], src substrate cortactin, transforming protein p21A, etc.) by 5-FU treatment in HeLa cervical carcinoma cells as determined by spot volume (P <0.05). Our experiments showed that 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of caspase-9 and caspase-3 as well as the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with an Apo-1/CD95 (Fas)-dependent fashion. In addition, we could observe reduction of HPV-18 E6/E7 gene expression and activation of p53, pRb, and p21waf1 proteins by 5-FU treatment in HeLa cervical carcinoma cells. In conclusion, we suggest that 5-FU suppresses the growth of cervical cancer cells not only by antiproliferative effect but also antiviral regulation. Our findings may offer new insights into the mechanism of anticancer effect affected by 5-FU treatment in cervical cancer cells and its mode of action.