Objectives: This study sought to understand the levels of vitamin K in the mature milk of Chinese lactating mothers, thereby providing a foundation for the development of appropriate intake (AI) of vitamin K for infants aged 0–5 months. Methods: Five hundred healthy lactating mothers were selected from the mature milk bank established by the Chinese Maternal and Infant Nutrition and Health Cohort by using a simple random sample procedure. Relevant information about lactating mothers and their infants was obtained by a questionnaire survey. Vitamin K1 and vitamin K2 (MK-4 and MK-7) in mature milk were determined by liquid chromatography–tandem mass spectrometry. Results: The total concentration of vitamin K in mature milk was 4.50 (2.85–6.33) ng/mL. The concentrations of vitamin K1, vitamin K2, MK-4, and MK-7 were 2.81 (1.66–4.39) ng/mL, 1.37 (0.75–2.11) ng/mL, 1.20 (0.58–1.97) ng/mL, and 0.13 (0.08–0.19) ng/mL, respectively. The concentration of vitamin K1 was highest and the concentration of MK-7 was lowest. The concentrations of vitamin K2 and MK-4 in mature milk from the south were significantly higher than those in mature milk from the north. The total vitamin K, vitamin K2, and MK-4 concentrations in mature milk of lactating mothers residing in urban areas were higher than those in rural areas. There was a tendency for the concentration of total vitamin K and vitamin K1 to increase with the mother’s age. Moreover, the concentration of MK-4 in mature milk was highest in summer, followed by spring and winter. The levels of vitamin K1 and MK-4 in mature milk were found to be affected by lactation stage; they were highest at 91–120 days and lowest at 31–60 days. Conclusions: Based on the concentration of vitamin K in mature milk found in this study, it is recommended that the appropriate intake of VK for Chinese infants aged 0–5 months is 3.49 μg/d (2.18 μg/d for VK1 and 1.06 μg/d for VK2).