PhexL222P mouse is a new ENU mouse model for XLH disease due to Leu to Pro amino acid modification at position 222. PhexL222P mouse is characterized by growth retardation, hypophosphatemia, hypocalcemia, reduced body bone length, and increased epiphyseal growth plate thickness and femur diameter despite the increase in PHEXL222P expression. Actually, PhexL222P mice show an increase in Fgf23, Dmp1, and Mepe and Slc34a1 (Na-Pi IIa cotransporter) mRNA expression similar to those observed in Hyp mice. Femoral osteocalcin and sclerostin and Slc34a1 do not show any significant variation in PhexL222P mice. Molecular dynamics simulations support the experimental data. P222 might locally break the E217-Q224 β-sheet, which in turn might disrupt inter-β-sheet interactions. We can thus expect local protein misfolding, which might be responsible for the experimentally observed PHEXL222P loss of function. This model could be a valuable addition to the existing XLH model for further comprehension of the disease occurrence and testing of new therapies.