Colorectal cancer is one of the leading causes of cancer mortality and morbidity worldwide. Previous studies indicate that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) positively regulates proliferation of intestinal epithelial cells and colorectal cancer cells. Importantly, inhibition of KLF5 expression in intestinal epithelial cells and colorectal cancer cells by pharmacologic or genetic means reduces their rate of proliferation. To identify additional and novel small molecules that inhibit KLF5 expression and thus colorectal cancer proliferation, we developed a reporter assay using colorectal cancer cell line (DLD-1) that stably expressed a luciferase reporter gene directed by 1,959 bp of the human KLF5 promoter upstream of the ATG start codon and performed a cell-based high-throughput screen with the Library of Pharmacologically Active Compounds that contains 1,280 biologically active compounds. The screen identified 8 potential inhibitors and 6 potential activators of the KLF5 promoter. Three potential inhibitors, wortmannin, AG17, and AG879, were further evaluated by secondary analyses. All three significantly reduced both KLF5 promoter-luciferase activity and protein level in DLD-1 cells in a dose- and time-dependent manner when compared with controls. They also significantly reduced the rate of proliferation of DLD-1 and two other colorectal cancer cell lines, HCT116 and HT29. Our results show the principle of using high-throughput screening to identify small-molecule compounds that modulate KLF5 activity and consequently inhibit colorectal cancer proliferation.