Colorectal cancer (CRC) may be particularly amenable to gene therapy because CRCs exhibit constitutive upregulation of Wnt signaling. We have previously demonstrated that butyrate, found in the colonic lumen, modulates Wnt signaling and nonspecifically upregulates transcription from minimal promoters. Because both of these actions may influence the efficiency and specificity of Wnt-targeted expression, the effects of butyrate on Wnt-targeted gene therapy were determined. Lithium is another agent known to upregulate Wnt activity in HCT-116 CRC cells and therefore may induce Wnt-targeted CRC cell kill. CRC cells were cotransfected with an expression vector for green fluorescent protein (GFP) and different versions of vectors coupling Wnt-sensitive promoters to FADD or diphtheria toxin A-chain (DT) effector genes. Cells were treated with butyrate and/or lithium chloride and flow cytometry was used to determine the percentage of remaining transfected (GFP-positive) cells. We demonstrate that promoter and cell type-specific differences occur in Wnt-specific cell kill induced by FADD and DT. A Wnt-sensitive version of the CMV promoter (TcfCMV) exhibited the optimum combination of efficient SW620 CRC cell kill and Wnt specificity; in addition, treatment with a physiologically relevant concentration of butyrate enhanced cell kill induced by TcfCMV-FADD while maintaining specificity. In HCT-116 CRC cells, optimum results were achieved utilizing TcfFos-DT constructs and cotreatment with both butyrate and lithium. The findings suggest that effective CRC cell kill can be achieved by gene therapy through modulation of Wnt signaling by butyrate and/or lithium.