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Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21^Cip/WAF induction via the extracellular signal–regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21^Cip/WAF by 150 nmol tautomycetin. The crucial role of p21^Cip/WAF1 in the extracellular signal–regulated kinase pathway–dependent antiproliferative responses by tautomycetin was confirmed by using p21^Cip/WAF1 gene–deleted HCT-116 cells. The growth inhibitory effect of …