Like normal colorectal epithelium, colorectal carcinomas (CRCs) are organized hierarchically and include populations of cells with stem-like properties. Leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) is associated with these stem cells in normal colorectal epithelium; however, the precise function of LGR5 in CRC remains largely unknown. Here, we analyzed the functional and molecular consequences of short hairpin RNA-mediated silencing of LGR5 in CRC cell lines SW480 and HT-29. Additionally, we exposed Lgr5-EGFP-IRES-CreERT2 mice to azoxymethane/dextrane sodium sulfate (AOM/DSS), which induces inflammation-driven colon tumors. Tumors were then flow-sorted into fractions of epithelial cells that expressed high or low levels of Lgr5 and were molecularly characterized using gene expression profiling and array comparative genomic hybridization. Silencing of LGR5 in SW480 CRC cells resulted in a depletion of spheres but did not affect adherently growing cells. Spheres expressed higher levels of several stem cell-associated genes than adherent cells, including LGR5. Silencing of LGR5 reduced proliferation, migration and colony formation in vitro and tumorigenicity in vivo. In accordance with these results, NOTCH signaling was downregulated upon LGR5 silencing. In AOM/DSS-induced colon tumors, Lgr5 high cells showed higher levels of several stem cell-associated genes and higher Wnt signaling than Lgr5 low tumor cells and Lgr5 high normal colon cells. Array comparative genomic hybridization revealed no genomic imbalances in either tumor cell fraction. Our data elucidate mechanisms that define the role of LGR5 as a marker for stem-like cells in CRC.