Increasing evidence suggests that carotenoids play an important role in visual and cognitive development during early life. This study aimed to depict the carotenoid profile in maternal/cord plasma and breast milk in three northern cities of China while investigating the association between dietary carotenoid intake and breast milk carotenoid levels. We enrolled 990 lactating mothers from three urban northern Chinese cities to collect breast milk (including colostrum, transitional milk, early mature milk, middle mature milk, and late mature milk). Among them, 90 participants also provided maternal/cord blood. The dietary carotenoid intake was recorded in the corresponding stages according to the Food Frequency Questionnaire and 24-hour dietary recall. The levels of carotenoids (lutein, zeaxanthin, β-cryptoxanthin, β-carotene, and lycopene) in maternal/cord plasma and breast milk were analyzed using high-performance liquid chromatography. We found a significant correlation between carotenoid concentrations in maternal and cord plasma. Moreover, carotenoid concentrations in maternal plasma were significantly higher than those in cord plasma. The total carotenoid levels in breast milk decreased from colostrum to late mature milk. β-Carotene predominated in colostrum, whereas lutein dominated in transitional and mature milk. We also found that breast milk carotenoid levels correlated with dietary carotenoid intake to a certain extent, particularly during the early stages of lactation. Therefore, we encourage women to appropriately increase their dietary intake of carotenoid-rich vegetables and fruits during pregnancy and lactation, particularly during work hours after lactation leave. Moreover, the variance of the carotenoid profile in maternal/cord plasma and breast milk across the three cities implied the necessity of further depicting the carotenoid status and influencing factors across different areas in China.