The aim of the present study was to determine the concentrations of estradiol (E2), estriol (E3) and progesterone in breast milk over different lactation periods, and to assess their relationship with the mother's diet. Ninety-six breast milk samples as well as 24-hour dietary records from 32 lactating mothers were collected on day 1 (colostrum), day 14 (transitional milk) and day 42 (mature milk) after delivery in Hangzhou, China. The concentrations of E2, E3 and progesterone differed significantly through different periods of lactation (p < 0.001). The content of E2 in colostrum, transitional milk and mature milk was 1.60 ± 0.96 μg L-1, 0.83 ± 0.36 μg L-1 and 1.26 ± 0.48 μg L-1, respectively. The concentrations of E3 were 2.09 ± 1.66 μg L-1, 2.23 ± 1.74 μg L-1 and 4.64 ± 2.15 μg L-1, respectively. The concentrations of progesterone were 6.10 ± 8.30 μg L-1, 4.25 ± 4.76 μg L-1 and 1.70 ± 2.42 μg L-1, respectively. The concentration of progesterone in breast milk was significantly negatively correlated with the intake of protein (p = 0.015), fat (p = 0.008), vegetables (p = 0.012), and meat and eggs (p = 0.036), while the concentration of E3 was significantly positively correlated with the intake of soy products (p = 0.025). This information indicates that the concentrations of E2, E3 and progesterone in breast milk varied over the lactating period. Dietary intake will to some extent affect the contents of E3 and progesterone in breast milk.