Simple SummaryEstrogen receptor-positive (ER+) breast cancer is the most prevalent breast cancer subtype, accounting for 70–85% of all cases. The combination of endocrine therapy, such as aromatase inhibitors (AIs), with target therapy is one of the most recent approaches, but its effectiveness is not optimal. Cannabidiol (CBD) has demonstrated important anti-tumor effects on ER+breast cancer cells. Considering this, our goal was to evaluate the effects of combining CBD with the AIs currently in use in the clinical context. Our results revealed that CBD may be particularly beneficial when combined with the AI exemestane (Exe), since it potentiates the anti-tumor effects of Exe through the modulation of cell death and specific targets, including ERα and androgen receptor (AR). This reinforces the beneficial potential of cannabinoids in breast cancer and points to the possibility of improving Exe effects through an adjuvant therapy with CBD.AbstractBackground: Estrogen receptor-positive (ER+) breast cancer is the most diagnosed subtype, with aromatase inhibitors (AIs) being one of the therapeutic drug types used in the clinic. However, endocrine resistance may develop after prolonged treatment, and different approaches, such as combining endocrine and targeted therapies, have been applied. Recently, we demonstrated that cannabidiol (CBD) induces anti-tumor actions in ER+breast cancer cells by targeting aromatase and ERs. Considering this, we studied, in vitro, whether CBD when combined with AIs could improve their effectiveness. Methods: MCF-7aro cells were used and the effects on cell viability and on the modulation of specific targets were investigated. Results: CBD when combined with anastrozole (Ana) and letrozole (Let) caused no beneficial effect in comparison to the isolated AIs. In contrast, when combined with the AI exemestane (Exe), CBD potentiated its pro-cell death effects, abolished its estrogen-like effect, impaired ERα activation, and prevented its oncogenic role on the androgen receptor (AR). Moreover, this combination inhibited ERK1/2activation, promoting apoptosis. The study of the hormonal microenvironment suggests that this combination should not be applied in early stages of ER+breast tumors. Conclusions: Contrary to Ana and Let, this study highlights the potential benefits of combining CBD with Exe to improve breast cancer treatment and opens up the possibility of new therapeutic approaches comprising the use of cannabinoids.