Mithramycin is an inhibitor of the binding of the Sp-family transcription factor to the GC box. Many studies show that mithramycin may reduce the expression of many oncogenes by inhibiting the mRNA and protein synthesis and it has been used as an antibiotic chemotherapy drug for a long time. Recently, Eps8 (EGFR pathway substrate 8) has been revealed to be a novel proto-oncogene related to cellular transformation, Rac activation and actin barbed-end-capping activity. Therefore, the aim of this study was to verify whether Eps8 might be regulated by mithramycin. Results showed that mithramycin could reduce the mRNA and protein levels of Eps8 in dose- and time-dependent manners in several cancer cell lines. Furthermore, cell growth and migration ability were also reduced significantly by mithramycin treatment. Since Src is a well-known Eps8 activity enhancer, a v-Src transfected IV5 cell line was subjected to mithramycin treatment and then analyzed to show that Src expression was unable to restore the mithramycin-induced decrease in Eps8 expression, cell growth, and migration ability. To further confirm the above mentioned results, the expression of Eps8 was eliminated by a transient transfection with siRNA and subsequent analysis showed that silencing of Eps8 might also lead to a reduced growth and migration ability of cancer cells. These findings suggested that Eps8 was involved in the regulation of growth and motility of cancer cells and mithramycin might exert its anticancer ability via a pathway involving the downregulation of Eps8.