A decline of estrogen in menopause women is accompanied with increases in many pro-inflammatory cytokines and osteoporosis. Andrographolide (AP), from Andrographis paniculata, which has an anti-inflammatory activity, may have potential to alleviate osteoporosis during estrogen deficiency. Here we report the promoting effects of AP on the differentiation of mouse pre-osteoblastic (MC3T3-E1) cells by increasing the expression and activity of alkaline phosphatase (ALP), an osteoblastic gene-specific marker. AP also accelerated bone formation and increased bone structural gene production including collagen and osteocalcin. We demonstrate for the first time the promoting effect of AP on the differentiation of osteoblast involved with the OPG/RANKL signaling pathway. AP also protected bone loss in the estrogen-deficient (ovariectomized, OVX) rats after 12 weeks of treatment. It protected the loss of bone mineral density, bone microarchitecture, and improved bone turnover rate in OVX rats. This study provides an essential evidence for clinical applications and development of AP towards treating osteoporosis in post-menopausal women.