A sensitive liquid chromatography-tandem mass spectrometric（LC-MS/MS) method was developed for the determination of pyridostigmine bromide in feed.The sample was extracted with acetonitrile or methanol，then concentrated to dryness under nitrogen，finally dissolved in water.The extract was purified with n hexane and solid phase extraction（SPE) column，then separated on a C18（150 mm×30 mm，3 μm) column by gradient elution using acetonitrile-5 mmol/L ammonium formate solution as mobile phase at a flow rate of 03 mL/min and a column temperature of 40 ℃.The qualitative analysis was performed with electrospray ionization in positive mode（ESI+) under multiple reaction monitoring（MRM) mode，and the quantitation was carried out by the isotope internal standard method.Results indicated that the calibration curves for pyridostigmine bromide in different feed matrixes showed good linear relationships in the concentration range of 1-100 μg/L with their correlation coefficients all above 0999.The limits of detection and quantitation were 10 μg/kg and 20 μg/kg，respectively.The recoveries ranged from 961% to 108% with relative standard deviations（RSDs) of 28%-83%.The method is sensitive and accurate，and is suitable for the determination of pyridostigmine bromide in compound feed，concentrated feed，additive premix and concentrate supplement.