As HER2 is frequently overexpressed in various malignancies, targeting HER2 is considered an efficient, highly selective antitumor therapy. HER2-targeted immunoconjugates are being developed and result in persistent remission of HER2-overexpressing tumors. However, many of the antibodies used as the targeting moiety are of murine origin and exhibit risk of inducing immunogenicity, limiting their antitumor therapeutic efficacy. Here, we humanized e23sFv, an HER2-targeting murine scFv with excellent affinity and specificity, using a human antibody consensus sequence engraftment strategy. The affinity of the initially humanized e23sFv was then rescued and improved by selective mutagenesis followed by phage-display-based affinity panning of the mutant pool. The resulting humanized e23sFv candidates (husFvs) exhibited up-to-94-fold increased affinity to recombinant HER2. The immunogenicity of e23sFv was dramatically alleviated after humanization, as indicated by the impaired production of cytokines by husFv-stimulated human PBMCs. Two internalizable husFvs with optimal affinity were applied to generate humanized immunoapoptotins by infusion with the translocation domain Fdt and the proapoptotic domain truncated Bid. The husFv-immunoapoptotins demonstrated improved HER2-targeting and tumor-killing capacities in vitro and in vivo compared with the e23sFv-immunoapoptotins and would enable the administration of multiple treatment cycles to patients, resulting in improved antitumor efficacy. Furthermore, the husFvs recognized distinct HER2 epitopes and could thus be used in combination with trastuzumab or pertuzumab to achieve robust synergistic antitumor effects in HER2-positive malignancies.