CK2 is a pleiotropic protein kinase, which phosphorylates many substrates and has a global role in promoting cell survival and preventing apoptosis. In this study, we investigated its involvement in the phenomenon of the drug resistance, by which tumor cells frequently become unresponsive to chemical apoptosis. By comparing the expression of CK2 subunits in four different pairs of sensitive (S) and resistant (R) cancer cell lines, we found that in three cases the resistant phenotype is accompanied by the overexpression of the CK2 catalytic alpha subunit, either alone or in combination with the regulatory beta subunit. The degree of CK2 expression correlates with the CK2 catalytic activity, when measured toward endogenous protein substrates. All the tested R cell lines, including the one with no CK2 overexpression, can be induced to undergo death by treatment with CK2 inhibitors. We therefore conclude that, although CK2 overexpression is not an absolute requirement for the resistant phenotype, its activity is essential for cell survival and contributes to a high degree of resistance. We also found that CK2 inhibition increases the accumulation of cytotoxic drugs inside the R cells, presumably by impairing the functionality of the extrusion pump P-gp. We therefore propose that CK2 should be considered a target to counteract the pharmaco-resistant phenotype.