Euphorbia ebracteolata was a natural medicine for the treatment of tuberculosis. The present work has performed the investigation of bioactive chemical substances from the roots of E. ebracteolata. Using various chromatographic techniques, 15 compounds were obtained from the roots of E. ebracteolata. On the basis of widely spectroscopic data analyses, the isolated compounds were determined to be diterpenoids, including rosane derivatives (1-12), isopimarane (13), abietane (14), and lathyrane (15), among which compounds 1-4, and 9 were undescribed previously. The inhibitory effects of isolated diterpenoids against Mycobacterium tuberculosis were evaluated using an Alamar blue cell viability assay. And two rosane-type diterpenoids 3 and 8 displayed moderate inhibitory effects on with the MIC values of 18 mu g/mL and 25 mu g/mL, respectively. For the potential inhibitor 3, the inhibitory effect against the target enzyme GImU was evaluated, which displayed a moderate inhibitory effect with the IC50 12.5 mu g/mL. Therefore, the diterpenoids from the roots of E. ebracteolata displayed anti-tuberculosis effects, which would be pay more attentions for the anti-tuberculosis agents. (C) 2018 Elsevier Inc. All rights reserved.