Disulfiram has been used in the treatment of alcoholism and exhibits an anti-tumor effect. However, the intracellular mechanism of anti-tumor activity of Disulfiram remains unclear. In this study, we focused on the modulatory role of Disulfiram via oncogenic factor carbonic anhydrase CA12 and its associated transporter anion exchanger AE2 in lung cancer cell line A549. The surface expression of CA12 and AE2 were decreased by Disulfiram treatment with a time-dependent manner. Disulfiram treatment did not alter the expression of Na+-bicarbonate cotransporters, nor did it affect autophagy regulation. The chloride bicarbonate exchanger activity of A549 cells was reduced by Disulfiram treatment in a time-dependent manner without change in the resting pH level. The expression and activity of AE2 and the expression of CA12 were also reduced by Disulfiram treatment in the breast cancer cell line. An invasion assay and cell migration assay revealed that Disulfiram attenuated the invasion and migration of A549 cells. In conclusion, the attenuation of AE2 and its supportive enzyme CA12, and the inhibitory effect on cell migration by Disulfiram treatment in cancer cells provided the molecular evidence supporting the potential of Disulfiram as an anticancer agent.