HM-3, an RGD (Arg-Gly-Asp)-modified antitumor polypeptide designed independently, has been demonstrated for its robust inhibitory effects on tumors. However, the intravenous administration and short half-life in vivo are inconvenient to its clinical application. To solve these issues, PEGylated HM-3 (mPEG-SC20k-HM-3) with prolonged half-time in vivo and subcutaneous administration was obtained after repeated screening of different types of PEG and numerous efficacy assays. The present study aimed to evaluate the antitumor activity and investigate the mechanism of the modified peptide to interpret the antitumor properties of mPEG-SC20k-HM-3 comprehensively and clearly. The results of the antitumor activity assays in vitro indicated that mPEG-SC20k-HM-3 exhibited a marked inhibitory activity on tumor metastasis and angiogenesis. mPEG-SC20k-HM-3 (73.4 mg/kg, sc) achieved a tumor inhibitory rate of 70.1% in an H460 (human non-small cell lung cancer) xenograft model with scarce cytotoxicity, compared with a rate of 72.2% for Avastin (10.0 mg/kg, iv). The mechanistic study showed that mPEG-SC20k-HM-3 could target integrin αvβ3 to block the downstream ERK and Akt pathways, as the expression levels of VEGF, Akt1, p-Akt1, ERK1/2, p-ERK1/2, MEK1, p-MEK1, integrin αv and β3 were reduced after HUVECs were incubated with mPEG-SC20k-HM-3 for 24 h. In conclusion, the antitumor activity of mPEG-SC20k-HM-3 in vitro and in vivo is promising and the mechanism was clearly reflected in the present study.