Human milk is considered as an optimal nutrition source for neonates, and vitamins A and E improve oxidative stress in offspring through breast milk. We aim to determine α-tocopherol and retinol contents in breast milk obtained over the course of lactation stages from mothers in three regions in China, and to investigate their correlation with maternal dietary intake during lactation. Human milk samples and 24 hour food records were obtained from mothers in Hangzhou (n = 34), Lanzhou (n = 34) and Beijing (n = 34) on day 1 (colostrum), day 14 (transitional milk) and day 42 (mature milk) postpartum. Concentrations of α-tocopherol and retinol in breast milk samples were measured by high-performance liquid chromatography. The breast milk concentrations of α-tocopherol and retinol decreased over progress of lactation stages (P < 0.001). The retinol content of transitional milk from Hangzhou was higher than that in Lanzhou (P = 0.019) and Beijing (P < 0.001), and the α-tocopherol content in colostrum from Hangzhou showed a lower concentration than that from Lanzhou (P = 0.001). There was a significantly positive correlation between retinol and α-tocopherol contents in colostrum (r = 0.37, P = 0.002), transitional (r = 0.41, P < 0.001) and mature milk (r = 0.53, P < 0.001). No significant correlation was found between any dietary constituents (grain, fruit, vegetable, dairy, egg and meat, oil and bean products) and either retinol or α-tocopherol.