Abstract Recent studies have implicated the DNA cytosine deaminase APOBEC3B as a major source of mutation in breast cancer. APOBEC3B explains a large proportion of both dispersed and clustered cytosine mutations, the latter of which are also called kataegis. APOBEC3B expression levels correlate with poor outcomes for patients with estrogen receptor positive breast cancer. While targeted therapies, such as tamoxifen, are available to treat these tumors, secondary drug resistance often develops. Here we suppressed endogenous APOBEC3B in the estrogen receptor positive breast cell line MCF-7L with shRNA. Lowered levels of APOBEC3B did not affect in vitro growth or sensitivity to estradiol. In a xenograft model of tamoxifen therapy, suppression of APOBEC3B associated with prolonged responses to tamoxifen (p<0.05). Furthermore, APOBEC3B over-expression did not affect in vitro cell growth but accelerated the development of tamoxifen-resistant tumors in vivo. In addition, we studied two separate cohorts of 285 breast cancer patients who received first line treatment with tamoxifen for recurrent disease. High APOBEC3B expression levels measured in the primary tumor associated significantly with unfavorable progression free survival in multivariate analysis that included the traditional predictive factors (age, dominant relapse site, disease-free interval, estrogen receptor and progesterone receptor, and adjuvant chemotherapy; HR=1.67, p=0.0001). The median period of progression free survival was 7.5 months for patients with APOBEC3B high primary tumors and 13.3 months for those with APOBEC3B low tumors (p<0.0.0001). These studies demonstrate that APOBEC3B drives resistance to endocrine treatment with tamoxifen in recurrent disease. Citation Format: Harris R, Law E, Sieuwerts A, LaPara K, Leonard B, Starrett G, Temiz NA, Sweep F, Span P, Foekens J, Martens J, Yee D. Tamoxifen resistance driven by the DNA cytosine deaminase APOBEC3B in recurrent estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-07.