Recent studies have revealed that the epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) are overexpressed in various types of human tumors and are attractive targets for anticancer drugs. In the present study, the expression of EGFR and IGF-1R in esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues in a tissue microarray was firstly detected by immunohistochemical staining. In addition, their co-overexpression was observed in 48 out of 75 (64%) patients. Based on the findings, the antitumor activity of an EGFR/IGF-1R bispecific and enediyne-energized fusion protein EGF-LDP-IGF-AE, which we constructed recently by fusing two ligands (EGF and IGF-1) with an enediyne antibiotic lidamycin (LDM), on ESCC were evaluated. Binding assay indicated that the EGF-LDP-IGF protein bound to esophageal cancer cells, and then internalized into the cytoplasm. Invitro, the enediyne‑energized fusion protein EGF-LDP-IGF-AE exhibited extremely potent cytotoxicity to ESCC cells with IC50 values between 10-10 and 10-15mol/l. Invivo, EGF-LDP‑IGF-AE also markedly suppressed the growth of human KYSE450 xenografts by 75.1% when administered at 0.3mg/kg in a nude mouse model, and its efficacy was significantly higher than that of LDM (at maximum tolerated dosage) and mono-specific counterparts. In addition, EGF-LDP-IGF-AE arrested cell cycle progression and it concentration-dependently induced cell apoptosis as well as inhibited the activation of EGFR/IGF-1R and two major downstream signaling pathways (PI3K/AKT and RAS/MAPK). These data imply the potential clinical application of EGF-LDP-IGF-AE for ESCC patients with EGFR and/or IGF-1R overexpression.