To determine the medicinal properties of Pleurotus cornucopiae and Safflower Oil (PS), the osteoblast effect was investigated. PS can prevent estrogen deficiency-induced bone loss. It was taken the measurements of biochemical factors such as serum osteocalcin, alkaline phosphatase, IGF-1, and bone status. Forty adult female Sprague-Dawley rats were randomly assigned to four equal groups for 12 weeks of treatment: (1) sham operation (SHAM) + vehicle; (2) ovariectomy (OVX) + vehicle; (3) OVX + 17β-estradiol (25 μg/kg); (4) OVX + PS (300 mg/kg/d, PO). At the end of the experiment, bone turnover, and trabecular microarchitecture were assessed by micro-computed tomography (micro-CT), and histopathology, respectively. Ovariectomized rats experienced remarkable increases in global volumetric bone density, and trabecular microarchitecture deterioration. These OVX-induced pathological changes are reversible in that most of them could be mostly corrected upon 17β-estradiol treatment. PNS treatment significantly prevented bone mass loss and microarchitecture deterioration. Moreover, PS enhanced osteoblast activity but suppressed osteoclast turnover, as evidenced by decreased levels of serum C-terminal telopeptides of type I collagen and elevated levels of alkaline phosphatase. PS mitigates estrogen deficiency-induced deterioration of trabecular microarchitecture.