Breast milk fatty acid composition may be affected by the maternal diet during gestation and lactation. The influence of dietary and breast milk fatty acids on breast milk immune factors is poorly defined. We determined the fatty acid composition and immune factor concentrations of breast milk from women residing in river/lake, coastal and inland regions of China, which differ in their consumption of lean fish and oily fish. Breast milk samples were collected on days 3–5 (colostrum), 14 and 28 post-partum (PP) and analysed for soluble CD14 (sCD14), transforming growth factor (TGF)-β1, TGF-β2, secretory IgA (sIgA) and fatty acids. The fatty acid composition of breast milk differed between the regions and with time PP. The concentrations of all four immune factors in breast milk decreased over time, with sCD14, sIgA and TGF-β1 being highest in the colostrum in the river and lake region. Breast milk DHA and arachidonic acid (AA) were positively associated, and γ-linolenic acid and EPA negatively associated, with the concentrations of each of the four immune factors. In conclusion, breast milk fatty acids and immune factors differ between the regions in China characterised by different patterns of fish consumption and change during the course of lactation. A higher breast milk DHA and AA concentration is associated with higher concentrations of immune factors in breast milk, suggesting a role for these fatty acids in promoting gastrointestinal and immune maturation of the infant.