Introduction: Despite recent advances in therapy, additional options for improving the outcomes of patients with HER2-overexpressing breast cancer (BC) are needed. The role of vitamin D in relation to BC outcomes is controversial. Both calcitriol and synthetic vitamin D analogues have been shown to increase sensitivity to chemotherapy in BC cell lines. In this study, we aimed to determine the effect of combined treatment with either calcitriol or the vitamin D analogue paricalcitol plus doxorubicin or paclitaxel in HER2+ BC cells. Methods: Cell characterization was performed using western blot for estrogen receptor (ER) α, progesterone receptor (PR) A-B, HER2 receptor and vitamin D receptor (VDR) A-B. Flow cytometry was used to determine the quantitative expression of HER2 in an established breast cancer cell line known to express VDR (SKBR3 [ERα-, PR A-B -, HER2+]). The effects of calcitriol and paricalcitol, alone or in combination with doxorubicin or paclitaxel, were evaluated using a Sulforhodamine B growth assay. We calculated IC20 inhibitory concentrations by non-linear regression analysis using sigmoidal fitting of dose-response curves. Results are presented as the mean cell proliferation percentage (%) ± standard deviation. Statistical analyses were carried out using one-way ANOVA and the Holm-Sidak method. Results: Calcitriol, paricalcitol, doxorubicin and paclitaxel inhibited cell proliferation in a dose dependent manner. Calcitriol 100nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 30.9% ± 24.1, compared to 74.6% ± 6.6 with calcitriol alone (p = 0.003) and 86.6% ± 4.1 with doxorubicin alone (p < 0.001). Similarly, the combination of calcitriol 100nM plus paclitaxel 0.001 μM inhibited the proliferation of SKBR3 cells to 11.6% ± 10.4, compared to 77.5% ± 6.1 with calcitriol alone (p < 0.001) and 50.7% ± 8.4 with paclitaxel alone (p < 0.001). Paricalcitol 25nM in combination with doxorubicin 0.01 μM inhibited the proliferation of SKBR3 cells to 49.1% ± 25.2, compared to 72.3 ± 13.2 with paricalcitol alone (p = 0.21) and with doxorubicin alone (p = 0.05). Paricalcitol 10nM in combination with paclitaxel 0.001μM inhibited the proliferation of SKBR3 cells to 26.1% ± 23.1, compared to 72.3% ± 13.2 with paricalcitol alone (p = 0.05) and 43.2% ± 12.1 with paclitaxel alone (p =0.285). Conclusions: Simultaneous treatment of SKBR3 cells with calcitriol plus either doxorubicin or paclitaxel showed a cooperative growth-inhibiting effect when compared with each cytotoxic drug alone. The combination of paricalcitol with each of the cytotoxic agents also showed a trend towards a higher growth-inhibiting effect, but failed to reach statistical significance when compared with each drug alone. Although these results point towards the presence of a cooperative antiproliferative effect of Vitamin D compounds on HER2+ BC cells when combined with chemotherapy, the concentrations needed to reach such an effect were high, which could potentially limit their use in clinical practice. Citation Format: Mateos-Soria AS, Garcia-Becerra RA, Diaz-Nieto L, Ventura-Gallegos JL, Jacobo-Herrera N, Soto-Perez-de-Celis E, Zentella-Dehesa A, Chávarri-Guera Y. Inhibitory effects of calcitriol and the vitamin D analogue paricalcitol in combination with chemotherapy on the growth of HER2 overexpressing breast cancer cells. [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 P5-04-15.