The Hedgehog (HH) signaling pathway leads to activation of GLI, which transcriptionally regulate target genes. Regulated HH signaling activity is critical during embryogenesis while aberrantly activated HH signaling is evident in a variety of human cancers. Canonical HH signaling engages the transmembrane receptor Patched (PTCH) and the signaling intermediate Smoothened (SMO) to activate GLI1 and GLI2. In addition GLI1 and GLI2 are activated by non-canonical oncogenic signaling pathways to further drive HH-dependent survival. We have demonstrated in human colon carcinoma cells that inhibition of the RAS/RAF pathway by U0126 decreases p-ERK protein expression and also inhibits GLI-luciferase activity and GLI1 mRNA and protein levels. Of importance is the demonstration that targeting of SMO (using cyclopamine) has minimal effect on cell survival in comparison to the inhibition of GLI (using GANT61), which induced extensive cell death in 7/7 human colon carcinoma cell lines. Genetic inhibition of the function of GLI1 and GLI2 by transient transfection of the C-terminus deleted repressor GLI3R, reduced proliferation and induced cleavage of caspase-3 and cell death in HT29 cells, similar to the effects of GANT61. Mechanistically, downstream of GLI1 and GLI2 inhibition, γH2AX (a marker of DNA double strand breaks) expression was upregulated, and γH2AX nuclear foci were demonstrated in cells that expressed GLI3R. Activation of the ATM/Chk2 axis with co-localization of γH2AX and p-Chk2 nuclear foci were demonstrated following GLI1/GLI2 inhibition. GANT61 induced cellular accumulation at G1/S and early S with no further progression before cells became subG1, while cDNA microarray gene profiling demonstrated downregulation of genes involved in DNA replication, the DNA damage response, and DNA repair, mechanisms that are currently being pursued. These studies highlight the importance of targeting the GLI genes downstream of SMO for terminating HH-dependent survival, suggesting that GLI may constitute a molecular switch that determines the balance between cell survival and cell death in human colon carcinoma.