CD44v6 is overexpressed in a variety of cancers, rendering it a promising target for radio-immunotherapy (RIT). In this study, we have characterized a novel engineered recombinant monoclonal anti-CD44v6 antibody, AbN44v6, and assessed its potential for use in RIT using either 177Lu or 131I as therapeutic radionuclides. In vitro affinity and specificity assays characterized the binding of the antibody labeled with 177Lu, 125I or 131I. The therapeutic effects of 177Lu-AbN44v6 and 131I-AbN44v6 were investigated using two in vitro 3D tumor models with different CD44v6 expression. Finally, the normal tissue biodistribution and dosimetry for 177Lu-AbN44v6 and 125I-AbN44v6/131I-AbN44v6 were assessed in vivo using a mouse model. All AbN44v6 radioconjugates demonstrated CD44v6-specific binding in vitro. In the in vitro 3D tumor models, dose-dependent therapeutic effects were observed with both 177Lu-AbN44v6 and 131I-AbN44v6, with a greater significant therapeutic effect observed on the cells with a higher CD44v6 expression. Biodistribution experiments demonstrated a greater uptake of 177Lu-AbN44v6 in the liver, spleen and bone, compared to 125I-AbN44v6, whereas 125I-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for 177Lu-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for 131I-AbN44v6. The effective dose was in the order of 0.1 mSv/MBq for both labels. In conclusion, AbN44v6 bound specifically and with high affinity to CD44v6. Furthermore, in vitro RIT demonstrated growth inhibition in a CD44v6-specific activity-dependent manner for both radioconjugates, demonstrating that both 177Lu-AbN44v6 and 131I-AbN44v6 may be promising RIT candidates. Furthermore, biodistribution and dosimetric analysis supported the applicability of both conjugates for RIT. The CD44v6-specific therapeutic effects observed with radiolabeled AbN44v6 in the 3D tumor models in vitro, combined with the beneficial dosimetry in vivo, render AbN44v6 a potential candidate for RIT.