Immunogenic responses by protein therapeutics often lead to reduced therapeutic effects and/or adverse effects via the generation of neutralizing antibodies and/or antidrug antibodies (ADA). Mirror-image proteins of the variable domain of the heavy chain of the heavy chain antibody (VHH) are potential novel protein therapeutics with high-affinity binding to target proteins and reduced immunogenicity because these mirror-image VHHs (d-VHHs) are less susceptible to proteolytic degradation in antigen-presenting cells (APCs). In this study, we investigated the preparation protocols of d-VHHs and their biological properties, including stereoselective target binding and immunogenicity. Initially, we established a facile synthetic process of two model VHHs [anti-GFP VHH and PMP12A2h1 (monomeric VHH of caplacizumab)] and their mirror-image proteins by three-step native chemical ligations (NCLs) from four peptide segments. The folded synthetic VHHs (l-anti-GFP VHH and l-PMP12A2h1) bound to the target proteins (EGFP and vWF-A1 domain, respectively), while their mirror-image proteins (d-anti-GFP VHH and d-PMP12A2h1) showed no binding to the native proteins. For biodistribution studies, l-VHH and d-VHH with single radioactive indium diethylenetriamine-pentaacid (111In-DTPA) labeling at the C-terminus were designed and synthesized by the established protocol. The distribution profiles were essentially similar between l-VHH and d-VHH, in which the probes accumulated in the kidney within 15 min after intravenous administration in mice, because of the small molecular size of VHHs. Comparative assessment of the immunogenicity responses revealed that d-VHH-induced levels of ADA generation were significantly lower than those of native VHH, regardless of the peptide sequences and administration routes. The resulting scaffold investigated should be applicable in the design of d-VHHs with various C-terminal CDR3 sequences, which can be identified by screening using display technologies.