The growing global demand for safe, effective, and sustainable bone regeneration materials is accelerating the search for innovative alternatives to conventional grafts. Trans-forming fishery by-products into high-performance biomaterials offers a unique opportunity to address both clinical needs and environmental sustainability. In two previous studies, we developed a nano-hydroxyapatite biomaterial (nanoS-HAp) from Chilean salmon (Salmo salar) backbone waste using a modified, simplified, and cost-effective alkaline hydrolysis–calcination method. This sustainable process produced a highly crystal-line, Ca/P-balanced, nano-structured material with excellent physico-chemico-mechanical properties and superior in vitro biocompatibility and osteoconductivity compared to widely used synthetic and xenogeneic hydroxyapatites. Building on these findings, the present study provides the first in vivo evaluation of our salmon-derived hydroxyapatite (SAHA). A critical-sized, non-vascularized calvarial defect model was established in 21 Sprague Dawley rats, with systemic safety and local de novo bone regeneration assessed at 15-, 30-, and 60-days post-implantation. Hematological and biochemical profiles showed no significant deviations from control values, while radiographic and histological anal-yses revealed enhanced bone callus formation and defect closure, with outcomes comparable to leading commercial bovine and human grafts. These results confirm the biocompatibility, osteogenic efficacy, and cost-friendly potential of SAHA in vivo. By transforming fishery waste into a high-performance and implantable HAp, this malleable material represents a promising and sustainable alternative for bone regeneration and repair.