The methods of extraction, separation, and purification of zizyphusine, spinosin, and 6′′′-feruloylspinosin from Zizyphi Spinosi Semen ( ZSS) were investigated. From 31.2 g defatted ZSS, 47.7 mg zizyphusine, 57.8 mg spinosin, and 80.5 mg 6′′′-feruloylspinosin were obtained after ultrasonic extraction, purification with macroporous resin, separation by flash chromatography, and purification by high-pressure preparative chromatography. The purities of zizyphusine, spinosin, and 6′′′-feruloylspinosin were 98.6%, 98.2%, and 99.0%, respectively, and their yields 85.0%, 82.8%, and 83.0%. The methods provide stable samples of these compounds for further study of their physiological function and application.