Breast cancer is the most common malignant cancer in women. CYP24A1 expression regulates cellular response to vitamin D, which has antitumor effects against breast cancer. This study aimed to identify the correlation between CYP24A1 mRNA expression and prognosis of breast cancer. This study enrolled 1102 patients, including 1090 females and 12 males, from TCGA-BRCA cohort. The Cancer Genome Atlas database was used to study CYP24A1 mRNA expression in breast cancer, and Chi-squared tests were performed to test the correlation between clinical features and CYP24A1 expression. The prognostic value of CYP24A1 in breast cancer was assessed using Kaplan-Meier curves and Cox analysis. Low CYP24A1 expression was associated with age, molecular subtype, ER, PR, HER2, menopause status, N classification, vital status, overall survial and relapse-free survival. CYP24A1 presented a moderate diagnostic ability in breast cancer. Furthermore, low CYP24A1 expression was correlated with poor prognosis. CYP24A1 was an independent risk factor for breast cancer. CYP24A1 plays an important role in prognosis of breast cancer. CYP24A1 has the potential to be a biomarker, especially in predicting prognosis.