Here, we report on the synthesis and biological evaluation of a novel peptide-drug conjugate, P6-SN38, which consists of the EGFR-specific short cyclic peptide, P6, and the Topo I inhibitor SN38, which is a bioactive metabolite of the anticancer drug irinotecan. SN38 is attached to the peptide at position 20 of the E ring's tertiary hydroxyl group via a mono-succinate linker. The developed peptide-drug conjugate (PDC) exhibited sub-micromolar anticancer activity on EGFR-positive (EGFR+) cell lines but no effect on EGFR-negative (EGFR-) cells. In vivo studies have shown that this PDC specifically accumulates in EGFR+ non-small cell lung cancer (NSCLC) xenografts and presents superior anticancer activity compared to the EGFR-specific antibody cetuximab (Erbitux) and free SN38. The 10 mg/kg dose of P6-SN38 in a side-by-side EGFR+/EGFR- xenograft shows eradication of the EGFR+ tumor with good tolerance, but no inhibition of tumor growth of the EGFR- counterpart. The PDC examined in this study was proven to be highly efficient for NSCLC, broadening its utilization for targeted cancer therapy in EGFR overexpressed cancers.