Shikonin, a naphthoquinone pigment isolated from the Chinese herbal Zicao, has been shown to exhibit antioxidant and anticancer effects. In the present study, we investigated the antiproliferative and pro-apoptotic effects of shikonin on A431 cells and explored the underlying molecular mechanisms. In the present study, our results showed that shikonin significantly inhibited the growth of A431 cells in a concentration- and time-dependent manner, and caused cell cycle arrest by upregulation of p21 and p27, and downregulation of cyclins and cyclin-dependent kinases. In addition, shikonin evidently induced apoptosis due to decreasing Bcl-2 expression, increasing Bax expression, activating caspase and inactivating NF-κB, while pretreatment with a pan-caspase inhibitor Z-Asp-CH2-DCB abrogated shikonin-induced apoptosis. Moreover, EGF could significantly increase the NF-κB DNA-binding activity and reversed the shikonin-induced inactivation of NF-κB. As anticipated AG1478 (EGFR inhibitor) and Bay11-7082 (NF-κB inhibitor) blocked EGF-reversed the inactivation of NF-κB induced by shikonin. Our data also showed that EGF could evidently reverse the shikonin-induced decreases in cell viability and increases in apoptosis. Then, the NF-κB inhibitors such as Bay11-7082, SN50, Helenalin and the EGFR inhibitor AG1478 and its downstream inhibitor such as PI3K inhibitor LY294002 and STAT3 inhibitor Stattic dramatically blocked EGF-reversed decreases in cell viability and increases in apoptosis induced by shikonin. Collectively, our findings indicated that shikonin inhibited cell growth and caused cell cycle arrest of the A431 cells through the regulation of apoptosis. Moreover, these effects were mediated at least partially by suppressing the activation of the EGFR-NF-κB signaling pathways.