Background: Cyathula officinalis Kuan is widely used in the clinics for the treatment of blood stasis in China. Objective: To evaluate the improving blood rheology and anti-inflammatory properties of C. officinalis Kuan extract (CO) and its active fraction (ACO) on acute blood stasis model Wistar rats and characterize the correlative constituents. Materials and Methods: CO at 0.26, 0.53, and 1.04 g/kg and ACO at 0.38, 0.75, and 1.5 g/kg were administered to acute blood stasis model Wistar rats for 3 days. Whole blood viscosity, plasma viscosity, and the levels of interleukin-6 (IL-6), nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and cyclooxygenase-2 (COX-2) in the plasma were measured. HPLC-QTOF/MS/MS method was used to identify the major constituents of ACO; the properties of two representative components (cyasterone and chikusetsusaponin IV) from ACO on thrombin-induced human umbilical vein endothelial cells damage model were also assessed by the levels of thromboxane A2 (TXA2), endothelin (ET), malondialdehyde (MDA), COX-2, endothelial nitric oxide synthase (eNOS), and superoxide dismutase (SOD). Results: CO and ACO significantly reduced whole blood viscosity, plasma viscosity, and levels of IL-6, NO, TNF-α, and COX-2 in vivo. Forty compounds were identified from ACO, mainly as phytoecdysteroids and saponins. Cyasterone and chikusetsusaponin IV could significantly inhibit levels of TXA2, ET, MDA, and COX-2 and promote the activities of eNOS and SOD in vitro. Conclusion: CO and ACO possessed significant improving blood rheology and anti-inflammatory effects on acute blood stasis model rats and the representative components Cyasterone and chikusetsusaponin IV showed significant anti-inflammatory, antioxidant, and anticoagulant effects in vitro.