In vitro studies on pathophysiology and innovative treatments of many orthopaedic diseases, based on the investigations of cells from pathologic skeletal tissues, greatly improve basic knowledge of osteoporosis. Primary osteoblast (OB) cultures derived from osteopenic bone from different species (human, rat, sheep) were compared to assess the differences that should be taken into account when performing in vitro biocompatibiliy tests or investigating pharmacological and physical treatments. Primary OB were isolated from osteopenic patients and animals by well-established methods and their metabolism was assessed with or without 1,25(OH)2D3. The greatest significant differences were observed between rat and human cells both under basal conditions and after 1,25(OH)2D3 stimulation. In addition, the response to 1,25(OH)2D3 stimulation of OBs from osteopenic rats was significantly different from that of human and sheep OB cultures, in terms of NO, OC, IL-6, and TGF-beta1. Cells derived from osteopenic sheep behaved much more similarly to those from humans, except for a significant difference in terms of TGF-beta1 observed both under basal conditions and after stimulation.