Abstract: A method of reversed phase high performance liquid chromatography for the determination of trans-vitamin K₁ in health products was established. The sample (0.25 g) was weighted after grinding the vitamin K₁ supplement tablets into powder, and then dissolved and made its volume up to 50 mL brown volumetric flask by isopropanol. The supernatant was filtered by a 0.45 μm filter membrane and separated on a Phenyl column with the mixtures of methanol and 50 mmol·L^-1 acetic acid solution at different volume ratios as mobile phase for gradient elution. The results showed that under the above chromatographic conditions, the resolution (R_S) of cis- and trans-vitamin K₁ was 1.76, linearity ranges were 0.01-2 mg·L^-1 and 0.04-8 mg·L^-1, with detection limits (3S/N) of 0.032 mg·L^-1 and 0.074 mg·L^-1, respectively. Recovery test was made by adding standard solution on blank samples at 3 concentration levels, giving values of recovery of trans-vitamin K₁ and cis-vitamin K₁ in the ranges of 92.0%-95.2% and 90.0%-101%, and intra-day RSDs (n=6) of the determined values were in the ranges of 1.6%-2.2% for trans-vitamin K₁ and 1.4%-2.3% for cis-vitamin K₁. The inter-day RSDs (n=5) of the determined values ranged from 2.4% to 4.8% for trans-vitamin K₁ and from 2.6% to 4.6% for cis-vitamin K₁. The proposed method was used for the analysis of actual samples, and the detection amount of trans-vitamin K₁ was in the range of 75.36-80.12 μg·g^-1.