Shengmai injection, consisting of Panax ginseng, Radix ophiopogonis and Schisandra chinensis, is a widely used Chinese medicine for the treatment of various cardiovascular diseases. In this study, tissue distribution and excretion of its multiple active components including protopanaxatriol-type (Ppt-type) ginsenosides (ginsenoside Rg1, Re, Rf and Rg2), protopanaxadiol-type (Ppd-type) ginsenosides (ginsenoside Rb1, Rd and Rc), ophiopogonin (ophiopogonin D), and lignan (schisandrin, schisandrol B and schizandrin B) in rat after single intravenous administration of Shengmai injection were reported. Ppt-type ginsenosides exhibited quick and wide distribution from blood into tissues and were eliminated rapidly through biliary, urinary and fecal excretions. Ppd-type ginsenosides Rb1, Rd and Rc distributed quickly from blood to all tissues but exhibited slow elimination by biliary and urinary excretions. Ophiopogonin D was excreted into bile with no urinary and fecal excretion, indicating its elimination in the form of secondary metabolites. Schisandrin, schisandrol B and schizandrin B was found to distribute quickly from blood into most tissues and had accumulation in these tissues. Very low biliary, urinary and fecal excretion implied that lignan was mainly excreted in the form of their metabolites. This study produced a first hand in vivo tissue distribution and dynamic profiles of the active components of Shengmai injection, providing valuable information for drug development and clinical application of Shengmai injection.