Abstract Disclosure: Y. Hoshino: None. K. Okamoto: None. K. Hajime: None. K. Irie: None. S. Kimura: None. N. Hidaka: None. D. Hagiwara: None. M. Nangaku: Dr. Nangaku receives research support and honoraria from Kyowa Kirin.. N. Makita: None. T. Saito: None. N. Ito: The Graduate School of Medicine and Faculty of Medicine, University of Tokyo, where Dr. Ito is affiliated, is an endowment department supported by Kyowa Kirin. Background and Objective: The most common cause of congenital FGF23-related hypophosphatemic rickets/osteomalacia (FGF23rHR) is X-linked hypophosphatemic rickets/osteomalacia (XLH), which is caused by loss-of-function mutations in the PHEX gene. In contrast, acquired FGF23rHR is primarily caused by tumor-induced osteomalacia (TIO). However, the causative tumor, known as phosphaturic mesenchymal tumor (PMT), is undetectable in 27-45% of TIO patients. This study hypothesized an autoimmune mechanism as a novel etiology for acquired FGF23rHR without detectable PMT. Methods: Thirteen patients with acquired FGF23rHR without detectable PMT were included. Controls consisted of 18 TIO patients with detectable PMT, 9 XLH patients, and 10 patients with other endocrine diseases. The presence of autoantibodies against PHEX, DMP1, ENPP1, and FGFR1—proteins implicated in congenital FGF23rHR—was evaluated using a luciferase immunoprecipitation system (LIPS) assay. Constructs of the target antigens fused with NanoLuc luciferase were transfected into Cos7 cells to generate lysates. After reacting patient sera with protein A/G magnetic beads, antibody-bead complexes were incubated with lysates, and luminescence was measured to detect autoantibodies. Additionally, flow cytometry using HEK293 cells expressing PHEX was performed to confirm the presence of anti-PHEX autoantibodies. Results: The LIPS assay detected anti-PHEX autoantibodies in 4 of 13 patients with acquired FGF23rHR without detectable PMT, but not in the controls. Autoantibodies against DMP1, ENPP1, and FGFR1 were not detected in any patients. Flow cytometry confirmed the presence of anti-PHEX autoantibodies in the same 4 sera identified by the LIPS assay, with one additional patient testing positive only by flow cytometry. Based on comparisons of biochemical findings, including serum phosphate, serum alkaline phosphatase (ALP), and serum bone-specific ALP, the disease severity in the anti-PHEX antibody-positive group was milder than that in the TIO group and similar to that in the XLH group. Discussion: Anti-PHEX autoantibodies were specifically identified in patients with acquired FGF23rHR without detectable PMT. Biochemical similarities between the anti-PHEX autoantibody-positive and XLH groups suggest that these autoantibodies contribute to the pathogenesis of osteomalacia. It is highly plausible that the anti-PHEX autoantibodies inhibit PHEX, triggering FGF23-related hypophosphatemia through a mechanism similar to that of XLH. Furthermore, the autoantibodies detected only by flow cytometry may recognize a unique three-dimensional structure of PHEX expressed on the cell membrane. Conclusion: This study identifies a novel disease entity, acquired osteomalacia associated with autoantibodies against PHEX (autoimmune osteomalacia: AIO). Presentation: Sunday, July 13, 2025