Methotrexate (MTX), has been reported to cause bone loss and bone marrow (BM) microvascular (sinusoids) damage. Icariin, an herbal flavonoid, has been widely shown to be beneficial for treatment of many diseases including osteoporosis and cardiovascular dysfunction. In the present study, we aimed to examine the potency of icariin on reducing bone loss and the dilation/damage of BM sinusoids in rats caused by MTX treatment. Groups of young rats were treated with 5 daily MTX injections (0.75 mg/kg) with/without icariin oral supplementation until day 9 after the first MTX injection. Histological analyses revealed that MTX-induced bone loss was attenuated by the icariin supplementation. MTX-induced adiposity was also suppressed by the icariin supplementation. In addition, histological examination found a reduction of BM sinusoidal dilation in metaphysis of icariin + MTX- treated rats when compared with the MTX alone-treated rats, and RT-PCR analyses showed upregulated expression of endothelial cell marker CD31 in the metaphysis of icariin + MTX-treated rats. Furthermore, in vitro investigations with rat sinusoid endothelial cells (SECs) suggest that icariin can potentially enhance the survival against cytotoxic effect of MTX and promote their abilities of migration and tube formation potentially via production of nitric oxide. Taken together, our results suggest a potential role of icariin in attenuating MTX-induced bone loss and a direct and/or indirect role in reducing MTX-induced BM sinusoidal dilation/damage in rats. Therefore, icariin may has a potential role in preventing bone loss and promoting or maintaining angiogenesis in the bone marrow in rats during/following MTX treatment.