Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that is highly expressed in rapidly dividing intestinal epithelial cells. KLF5 binds to GC-rich sequences in promoters of numerous genes, including cyclin D1, cyclin B1/Cdc2, and integrin-linked kinase. KLF5 mediates the transforming effects of oncogenic H-Ras, and as a result it is thought to be important in regulating colon cancer pathogenesis, thus KLF5 inhibitors are of interest in cancer chemotherapy. We used an ultra-high throughput screening (uHTS) strategy to identify and confirm KLF5-specific activity and potency of Molecular Libraries Small Molecule Repository (MLSMR) compounds, also using a cytotoxicity counterscreen with KLF5-deficient IEC-6 colon cell line to eliminate false positives. The SRIMSC performed the KLF5 and IEC-6 assays in dose response format, validating selected HTS hits for re-purchase and for follow-up studies. We initiated both structure activity relationship (SAR)-by-purchase and in-house synthesis campaigns. The assay provider evaluated hits and their analogs to determine potency, selectivity, and mechanism of action, establishing the necessary assays for proper evaluation (e.g., western blotting, cytotox evaluation, and cell cycle studies; all data were submitted to PubChem). The team ultimately chose to focus efforts on a lead series which provided a compound fully meeting the probe criteria for KLF5 inhibition: ML264, also designated CID 51003603, SID 117686865, and SR-03000002171. ML264 is highly active (IC50 = 29 nM is a cell-based assay for proliferation of DLD-1 cells, IC50 = 81 nM in a cell-based luciferase assay). It lacks cytotoxicity in the IEC-6 control cell line (IC50 >50 μM, <50% inhibition was observed at 100 μM). Robust activity was also seen in several other KLF5-expressing cell types as well (e.g., HCT116, IC50 = 560 nM; HT29, IC50 = 130 nM; SW620, IC50 = 430 nM). ML264 does not inhibit kinases associated with the KLF5 pathway, as determined using a panel of 47 selected kinases. Western blot analysis shows that ML264 significantly reduces KLF5 expression. These results demonstrate KLF5 target specificity. An NCI60 panel study using ML264 revealed that it induces death of most colon cancer cell lines, with cytotoxicity toward several other tumor cell lines as well. ML264 is chemically stable, unreactive with glutathione, has suitable aqueous solubility, is highly stable to mouse, rat, and human hepatic microsomes, has favorable properties associated with drug-likeness, and does not inhibit CYP enzymes. Due to these properties in concert with its high cellular potency and selectivity, ML264 is a good candidate for in vivo anticancer studies, with great potential for use in long time-course in situ studies aimed to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium.