Abstract: LC-MS/MS was applied to the determination of vitamin D3 and 25-hydroxy vitamin D3 in poulty. After saponification,SPE purification and positive phase preparation of the sample,symmetry C18 chromatographic column was used for separation,and APCI and multi-reactions monitor were adopted in MS/MS. Deuterated vitamin D3 was used as internal standard. Linearity ranges of vitamin D3 and 25-hydroxy vitamin D3 were found in the same range of 5-100 μg·L-1 with same detection limits (3S/N) of 0.60 μg·kg-1. The proposed method was applied to the analysis of pork sample,giving values of recovery and RSD′s (n=6) in the ranges of 75.0%-97.1% and 1.9%-5.6%,respectively.