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Effects of the pulsed magnetic field on membrane fluidity of Bacillus subtilis were investigated. 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as fluorescence probe to label the membrane of B. subtilis at 35℃ for 45min to determine the changes of fluorescence polarization and fluorescence anisotropy of B. subtilis membrane by fluorescence polarization technique. The results indicated that fluorescence polarization and fluorescence anisotropy increased with the increase of intensity and pulse number of PMF. Compared with the control group, fluorescence polarization increased greatly at 2.0T with 25~30 pulses, 2.5T with 25~30 pulses, 3.0T with 20~30 pulses and 3.3T with 10~30 pulses（P＜0.05）. Fluorescence anisotropy also increased greatly at 2.0T with 30 pulses, 2.5T with 25~30 pulses, 3.0T with 20~30 pulses and 3.3T with 15~30 pulses（P＜0.05）. The membrane fluidity decreased, resulting in the lost of the membrane function, when treated by PMF, which suggested a perhaps mechanisms for the cell death.