Choline is an essential nutrient required for methyl group metabolism, but its role in carcinogenesis and tumor progression is not well understood. By utilizing a population-based study of 1508 cases and 1556 controls, we investigated the associations of dietary intake of choline and two related micronutrients, methionine and betaine, and risk of breast cancer. The highest quintile of choline consumption was associated with a lower risk of breast cancer [odds ratio (OR): 0.76; 95% confidence interval (CI): 0.58-1.00] compared with the lowest quintile. Two putatively functional single nucleotide polymorphisms of choline-metabolizing genes, PEMT -774G>C (rs12325817) and CHDH +432G>T (rs12676), were also found be related to breast cancer risk. Compared with the PEMT GG genotype, the variant CC genotype was associated with an increased risk of breast cancer (OR: 1.30; 95% CI: 1.01-1.67). The CHDH minor T allele was also associated with an increased risk (OR: 1.19; 95% CI: 1.00-1.41) compared with the major G allele. The BHMT rs3733890 polymorphism was also examined but was found not to be associated with breast cancer risk. We observed a significant interaction between dietary betaine intake and the PEMT rs7926 polymorphism (P(interaction)=0.04). Our findings suggest that choline metabolism may play an important role in breast cancer etiology.