Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate (12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decreases in superoxide dismutase and Na+-K+-ATPase activity, induced by glutamate. It also reduced the concentration of malondialdehyde, enhanced the mitochondrial transmembrane potential, inhibited the elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells. Glutamate application is an established method of inducing PC12 cell injury. PC12 cells were cultured with serum containing Tongqiaohuoxue decoction consisting of moschus, Carthamus tinctorius, Rhizoma chuanxiong, Semen pruni persicae, and Radix Paeoniae Rubra. After 24 hours of co-cultivation, glutamate 12.5 mM) was added to the culture medium. We found that serum containing Tongqiaohuoxue decoction prevented the increase in reactive oxygen species, and the decrease in superoxide dismutase and Na + -K + -ATPase activity, induced by glutamate. It also reduces the concentration of malondialdehyde , enhanced the mitochondrial transmembrane potential, inhibited elevation of cellular calcium, and decreased phosphorylation of calmodulin-dependent protein kinase II. Thus, serum containing Tongqiaohuoxue decoction had protective effects on cell proliferation and membrane permeability in glutamate-injured PC12 cells.