Inactivation of epidermal growth factor receptor (EGFR) family members are prime targets for cancer therapy. Here, we show that tephrosin, a natural rotenoid which has potent antitumor activities, induced internalization of EGFR and ErbB2, and thereby induced degradation of the receptors. Treatment of HT-29 cells with tephrosin inhibited both the ligand-induced and constitutive phosphorylation of EGFR, ErbB2 and ErbB3, and concomitantly suppressed the activation of the downstream signaling molecules such as Akt and Erk1/2 mitogen-activated protein kinase (MAPK). Tephrosin caused internalization of EGFR and ErbB2 into vehicles, which resulted in degradation of the receptors. This degradation was blocked by the lysosomal inhibitor, chloroquine. We also showed that tephrosin induced apoptosis. Tephrosin did not induce the proteolytic processing of caspase-3 and poly(ADP-ribose) polymerase (PARP), but did nuclear translocation of apoptosis-inducing factor (AIF), suggesting that tephrosin may induce caspase-independent apoptosis. These findings provide the first evidence that tephrosin could exert antitumor effects by inducing internalization and degradation of inactivated EGFR and ErbB2 in human colon cancer cells.