Background: 15,16-dihydrotanshinone I (DHTI), a lipophilic tanshinone extracted from Danshen root (Salvia miltiorrhiza Bunge), has been reported to function as an antitumor agent. However, its activity on osteosarcoma (OS), the most common primary malignant bone tumor, is unclear.Objective: This study aimed to determine the effects of DHTI treatment on proliferation, apoptosis and migration of human OS cell line 143B and investigate the possible underlying molecular mechanisms. Method: Human cell line 143B was used as a model for investigation of the inhibitory effects of DHTI on osteosarcoma. Cell proliferation was evaluated by MTT assays, while cell cycle progression, apoptosis and cell migration were analyzed by flow cytometer, caspase activity assays and scratch migration assays. qRT-PCR and western blot were carried out to detect the expression levels of representative genes and proteins during physiological processes examined above. Results: DHTI treatment inhibited the proliferation of 143B cells in a dose- and time-dependent manner through arresting cells in G1 phase by reducing the expression of cyclin D1, cyclin E1, CDK2, CDK4, CDK6, p-Rb, E2F1, SKP2 and increasing the expression of P53, P21cip1, P27kip1. In addition, DHTI induced apoptosis of 143B cells through caspase pathways to activate caspase-3, caspase-8, caspase-9, Bax, and PARP cleavage but reduce the expression of Bcl-2. Furthermore, DHTI treatment attenuated cell migration by down-regulating adhesion molecules VCAM-1 and ICAM-1. Conclusion: These findings suggest that DHTI could be a novel and efficient therapeutic candidate for OS treatment and further detailed investigation is warranted.