Monoclonal antibodies and peptides are conjugated to the surface of nanocarriers (NCs) for targeting purposes in numerous applications. However, targeting efficacy may vary with their specificity, affinity, or avidity when linked to NCs. The physicochemical properties of NCs may also affect targeting. We compared the targeting efficacy of the CD4 binding peptide BP4 and an anti‐CD4 monoclonal antibody (CD4 mAb) and its fragments, when conjugated to lipid‐coated poly(lactic‐co‐glycolic) acid nanoparticles (LCNPs). Negatively charged LCNPs with cholesteryl butyrate in the lipid layer (cbLCNPs) dramatically reduced nonspecific binding, leading to higher targeting specificity, compared to neutral or positively charged LCNPs with DOTAP (dtLCNP). cbLCNPs surface conjugated with a CD4 antibody (CD4‐cbLCNPs) or its fragments (fCD4‐cbLCNPs), but not BP4, showed high binding in vitro to the human T cell line 174xCEM, and preferential binding to CD3+ CD14‐CD8‐ cells from pigtail macaque peripheral blood mononuclear cells. CD4‐cbLCNPs showed 10‐fold higher binding specificity for CD4+ than CD8+ T cells, while fCD4‐cbLCNPs demonstrated the highest binding level overall, but only three‐fold higher binding specificity. This study demonstrates the importance of ζ‐potential on NC targeting and indicates that CD4 mAb and its fragments are the best candidates for delivery of therapeutic agents to CD4+ T cells. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1177–1188, 2018.