Allolobophora calignosa (Ac) is a folk medicine for millennia, as it possesses many biological activities. This study aimed to investigate the chemo-preventive activity of A.calignosa coelomic fluid (AcCF) and A.calignosa extract (AcE) on glucocorticoid-induced osteoporosis (GIOP) in mice. Characterization and in vitro biological activity of AcE and AcCF has been assessed. Male CD-1 mice were subcutaneously received dexamethasone (DEX) (1 mg/kg, 5 times/week) and concurrently intraperitoneally treated with either AcCF (20 mg/kg) or AcE (45 mg/kg) every other day for 28 days. Serum and bone homogenates were subjected for qPCR and biochemical analysis. AcE and AcCF treatment significantly increased bone mineral density (BMD), bone mineral content (BMC), calcium (Ca), phosphorus (P), and calcitonin levels, whereas activity of serum alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), serum acidic phosphatase (ACP), bone acidic phosphatase (BACP) and parathyroid hormone (PTH) levels were significantly reduced compare with untreated GIOP mice. Treatment with AcE and AcCF modulates oxidative stress and downregulated Rank and Mmp9 expression, as well as increased glycosaminoglycan content in the organic bone matrix, resulting in osteoclastogenesis inhibition. Overall, AcCF and AcE show a chemo-preventive activity against GIOP by inhibiting oxidative stress and regulating expression and/or activity of osteoblast/osteoclast-related markers.