After periprosthetic joint infection (PJI)-dependent revision surgery, a significantly elevated number of patients suffer from prosthesis failure due to aseptic loosening and require additional revision surgery despite clearance of the initial infection. The mechanisms underlying this pathology are not well understood, as it has been assumed that the bone stock recovers after revision surgery. Despite clinical evidence suggesting decreased osteogenic potential in PJI, understanding of the underlying biology remains limited. In this study, we investigated the impact of PJI on bone homeostasis in a two-stage exchange approach at explantation and reimplantation. Sixty-four human tibial and femoral specimens (20 control, 20 PJI septic explantation, and 24 PJI prosthesis reimplantation samples) were analyzed for their bone microstructure, cellular composition, and expression of relevant genetic markers. Samples were analyzed using X-ray microtomography, Alcian blue and tartrate-resistant acid phosphatase staining, and RT-qPCR. In patients with PJI, bone volume (BV/TV;​ 0.173 ± 0.026;​ p < 0.001), trabecular thickness (164.262 ± 18.841 μm;​ p < 0.001), and bone mineral density (0.824 ± 0.017 g/cm²;​ p = 0.049) were reduced;​ trabecular separation (1833.939 ± 178.501 μm;​ p = 0.005) was increased. While prevalence of osteoclasts was elevated (N.Oc/BS:​ 0.663 ± 0.102, p < 0.001), osteoblast cell numbers were lower at explantation (N.Ob/BS:​ 0.149 ± 0.021;​ p = 0.047). Mean expression of bone homeostasis markers osteocalcin, osteopontin, Runx2, TSG-6, and FGF-2 was significantly reduced at prosthesis explantation. Despite partial recovery, all analyzed parameters were still significantly impacted at reimplantation. In contrast, mean expression of osteoclastogenesis-stimulating cytokine IL-17a was significantly increased at both explantation and reimplantation. In this study, we found a strong and lasting impact of PJI on the bone homeostasis on a molecular, cellular, and microstructural level. These changes may be responsible for the increased risk of prosthesis failure due to aseptic loosening. Our data suggest there is significant potential in modulating bone homeostasis to improve prosthesis fixation and long-term clinical outcome in affected patients.