Antimicrobial silver nanoparticle coatings have attracted interest for reducing prosthetic joint infection. However, few studies report in vivo investigations of the biotransformation of silver nanoparticles within the regenerating tissue and its impact on bone formation. We present a longitudinal investigation of the osseointegration of silver nanoparticle-coated additive manufactured titanium implants in rat tibial defects. Correlative imaging at different time points using nanoscale secondary ion mass spectrometry, transmission electron microscopy (TEM), histomorphometry, and 3D X-ray microcomputed tomography provided quantitative insight from the nano- to macroscales. The quality and quantity of newly formed bone is comparable between the uncoated and silver coated implants. The newly formed bone demonstrates a trabecular morphology with bone being located at the implant surface, and at a distance, at two weeks. Nanoscale elemental mapping of the bone–implant interface showed that silver was present primarily in the osseous tissue and colocalized with sulfur. TEM revealed silver sulfide nanoparticles in the newly regenerated bone, presenting strong evidence that the previously in vitro observed biotransformation of silver to silver sulfide occurs in vivo.