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Few epidemiological studies have evaluated the association of choline and betaine intake with breast cancer risk and the results remain inconsistent. This study aimed to assess the relationship between dietary intake of choline and betaine and the risk of breast cancer among Chinese women. A two-stage case-control study was conducted, with 807 cases and 807 age- (5-year interval) and residence (rural/urban)-matched controls. A validated food frequency questionnaire was used to assess dietary intake by face-to-face interview. An unconditional logistic regression model was used to calculate multivariate-adjusted odds ratios (OR) and 95% confidence intervals (CI). A significant inverse association was found between dietary choline and betaine consumption and breast cancer risk. The adjusted OR for the highest quartile of intake compared with the lowest were 0.40 (95% CI = 0.28–0.57, Ptrend < 0.001) for total choline intake, 0.58 (95% CI = 0.42–0.80, Ptrend < 0.001) for betaine intake and 0.38 (0.27–0.53, Ptrend < 0.001) for choline plus betaine intake, respectively. Intakes of individual choline compouds, choline from glycerophosphocholine, phosphocholine, phosphatidylcholine, sphingomyelin and free choline were also negatively associated with breast cancer risk. The inverse association between choline intake and breast cancer risk was primarily confined to participants with low folate level (<242 g/day), with an OR (95% CI) of 0.46 (0.23–0.91) comparing the fourth quartile with the first quartile of choline intake (Ptrend = 0.005). The present study suggests that consumption of choline and betaine is inversely associated with the risk of breast cancer. The association of choline intake with breast cancer risk is probably modified by folate intake.