Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain aetiology. The therapeutic phenylalanine (Phe)-restricted diet and the possible noxious effects of high plasma Phe concentrations on bone have previously been suggested as possible determinant factors. Since osteoclasts are involved in bone reabsorption, they could play a role in determining bone damage in PKU. The reported increased excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although different diseases characterized by bone loss have been related to increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs), to date there is no evidence of increased osteoclast formation in PKU. In this study, we compared the spontaneous osteoclastogenesis from PBMCs in 20 patients affected by PKU with that observed in age- and sex-matched healthy subjects. Phenylketonuric patients showed the number of osteoclasts to be almost double that observed in controls (159.9 ± 79.5 and 87.8 ± 44.7, respectively; p = 0.001). Moreover, a strict direct correlation between the spontaneous osteoclastogenesis in PKU patients and the mean blood Phe concentrations in the preceding year was observed (r = 0.576; p = 0.010). An imbalance between bone formation and bone resorption might explain, at least in part, the pathogenesis of bone loss in this disease. These findings could provide new insights into the biological mechanisms underlying bone damage in PKU.