The combination of sodium butyrate (NaB) and ganciclovir (GCV) was considered to be a noteworthy therapeutic strategy in Epstein-Barr virus (EBV)-associated cancers. However, clinical studies have indicated that an extremely high dose of NaB is required to obtain the expected curative efficacy. This obviously limits the practical clinical application of the two drugs combined. In this study, we investigated the possibility of sensitizing tumor cells to NaB and GCV mediated cytotoxicity by modulating intracellular signal pathways. The results showed that the disruption of Ras/Raf activity by expressing dominant negative forms of both Ras and Raf-1 did not alter the potency of the NaB and GCV combination in the EBV-positive cell line, B95-8. However, blocking Akt activity by expressing its dominant negative form remarkably promoted NaB and GCV-mediated cytotoxicity via a thymidine kinase (TK)-independent mechanism. Interestingly, it was found that the constitutive activation of mitogen-activated protein kinase kinase kinase 1 (MEKK1) dramatically enhanced the sensitization of the cells to the combination of NaB and GCV, accompanied with an increase in TK expression in B95-8 cells. These results suggest that interfering with either the Akt or MEKK1 signaling pathway may be a useful therapeutic strategy to increase the sensitivity of EBV-positive tumor cells to the combination of NaB and GCV.