Objective: To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction (下瘀血汤, XYXD) on activation of hepatic stellate cells (HSCs) and defenestration of sinusoidal endothelial cells (SECs) in CCI4-induced fibrotic liver of mice. Methods: High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCI4 exposure and administered with XYXD for 6 weeks simultaneously. Liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. Sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 (CD31). Whole liver lysates were detected of α-smooth muscle actin (α-SMA) and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay and cellomics arrayscan, respectively. Results: Amygdalin and emodin in XYXD were identified. XYXD (993 mg/kg) inhibited Sirius red positive area up to 70.1% (P〈0.01), as well as protein levels of α-SMA and type- I collagen by 42.0% and 18.5% (P〈0.05) respectively. In vitro, XYXD (12.5 μg/mL, 50 μg/mL) suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% (P〈0.05). Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% (P〈0.05) and 49.5% (P〈0.01), evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 (P〈0.05). Conclusions: XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XY