<b><i>Introduction:</i></b> The present study aimed to explore the relationship between the dietary intake and the human milk (HM) fatty acid (FA) profile of Chinese lactating women. <b><i>Methods:</i></b> HM samples and food records were obtained from 122 Chinese women over 5 visits between 0 and 51 days postpartum. Adjusted multiple regression was performed to explore associations between maternal dietary intakes of energy, macronutrients, FAs and foods, and the HM FA profile. Analyses were performed separately for colostrum and mature milk. <b><i>Results:</i></b> Dietary intakes of total polyunsaturated FAs (PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid were positively associated with the HM contents of PUFAs, omega-6 (n-6) PUFAs, and linoleic acid (LA), and the intakes of n-3 PUFAs and α-linolenic acid (ALA) were negatively associated with saturated FA levels in HM. Associations were stronger for mature milk. Intakes of milk/dairy, meat/poultry, and eggs were negatively associated with n-6 PUFAs, LA, and EPA in mature milk, whereas the opposite was seen for fish/shrimp. Positive associations were also found between fish/shrimp and total and n-3 PUFAs in mature milk. <b><i>Conclusion:</i></b> The HM FA profile of Chinese women is associated with their diet, and in particular with their FA intake. Tailored nutritional advice based on HM FA composition may optimize HM FA profile and thereby contribute to healthy infant development.