Liver cancer is a worldwide threat to human health. High expression levels of C-X-C chemokine receptor type 4 (CXCR4) have been reported to promote the migration and invasion capacities of liver cancer cells. Cordycepin, extracted from Cordyceps militaris, has anti-inflammatory, antioxidant and anticancerous properties. Therefore, in the present study, migration assays, western blotting, reverse transcription-quantitative PCR and immunofluorescence analyses were conducted to determine whether cordycepin was able to suppress the migration and invasion abilities of liver cancer cells by inhibiting CXCR4 expression. The results suggested that cordycepin notably inhibited migration and invasion, and decreased the expression of CXCR4 in a dose-dependent manner. Activation of phosphorylated (p-) NF-κB inhibitor α (IκBα) and p-P65, the principal components of the NF-κB signaling pathway, was also downregulated. In addition, cordycepin markedly suppressed the nuclear translocation of P65, but had no effect on the expression of total IκBα (t-IκBα) and total P65 (t-P65). JSH-23, an inhibitor of the NF-κB pathway, impaired the migration of liver cancer cells, and was found to act synergistically with cordycepin. Furthermore, cordycepin treatment reduced the chemotactic migration ability of liver cancer cells to stromal cell-derived factor 1 (SDF1), which was significantly enhanced following treatment with JSH-23. Collectively, the present results indicated that cordycepin inhibited the nuclear translocation of P65 by preventing p-IκBα activation; this resulted in the downregulation of CXCR4 expression, and subsequently, in the impaired migration and invasion abilities of liver cancer cells and attenuated reactivity to SDF1. The current study revealed a novel mechanism for the antimetastatic activity of cordycepin and its potential to exert positive synergistic effects with other compounds for the treatment of liver cancer.