The hedgehog (Hh) signaling pathway has been shown to be activated in the cancer stem cells of several tumor entities. The Hh inhibitor GDC-0449 has been proven to be effective in some cancers but not yet in lung cancer. We aimed at investigating whether GDC-0449 is effective in the lung cancer cell lines HCC (adenocarcinoma) and H1339 (small-cell-lung carcinoma), whether in these cell lines stem cell-like side populations (SPs) can be identified, and whether possible effects of GDC-0449 are mediated via SPs. SPs were identified by spectrum shift and decreased fluorescence after staining with 2.5 μg/ml Hoechst 33342. Expression of proteins was quantified by immunofluorescence. GDC-0449 (25 and 50 μM) inhibited concentration-dependent cell growth in HCC and H1339 cells. Further, the inhibitory effects of cisplatin on cell growth were augmented. In HCC and H1339 cell lines, SPs of 0.57 and 0.46% could be identified, respectively. SP, but not non-SP, cells were able to repopulate the original tumor population. The Hh receptor smoothened was detectable in SP but not in non-SP cells, showing the activation of the Hh pathway only in SPs. GDC-0449 considerably reduced SPs in HCC and H1339 cells. We demonstrate for the first time that GDC-0449 effectively reduces cell growth in lung cancer cell lines. This effect is mediated by the inhibition of stem cell-like SPs.