Integrin alpha(5)beta(1) is expressed on several types of cancer cells, including colon cancer, and plays an important role in tumor growth and metastasis. The ability to target the integrin alpha(5)beta(1) using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth, reducing tumor metastasis, and decreasing deleterious side effects associated with different cancer therapies. Liposomes are nano-sized phospholipid bilayer vesicles that have been extensively studied as drug delivery carriers. The goal of this study is to design stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that will target colon cancer cells that express the integrin alpha(5)beta(1). The PEG provides a steric barrier allowing the liposomes to circulate in the blood and the functionalizing moiety, PR_b peptide, will specifically recognize and bind to alpha(5)beta(1) expressing cells. PR_b is a novel peptide sequence that mimics the cell adhesion domain of fibronectin, and includes four building blocks, RGDSP (the primary recognition site for alpha(5)beta(1)), PHSRN (the synergy site for alpha(5)beta(1)), a (SG)(5) linker, and a KSS spacer. In this study we have demonstrated that by varying the amount of PEG (PEG750 or PEG2000) and PR_b on the liposomal interface we can engineer nano-vectors that bind to CT26.WT, HCT116, and RKO colon cancer cells in a specific manner and are internalized through most likely alpha(5)beta(1)-mediated endocytosis. GRGDSP-targeted stealth liposomes bind to colon cancer cells and internalize, but they have much lesser efficiency than PR_b-targeted stealth liposomes, and more importantly they are not as specific since many integrins bind to RGD peptides. PR_b-targeted stealth liposomes are as cytotoxic as free 5-Fluorouracil (5-FU) and exert the highest cytotoxicity on CT26.WT cells compared to GRGDSP-targeted stealth liposomes and non-targeted stealth liposomes. Thus, the proposed targeted delivery system has the great potential to deliver a therapeutic load directly to colon cancer cells, in an efficient and specific manner.