miR-223 is an anti-inflammatory microRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer (BC), we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during BC onset and progression. miR-223 expression was decreased in BC of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells miR-223 acted cell autonomously in the control of their growth and morphology in 3D-context. In the MMTV-∆16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal BC resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from luminal and HER2-positive BC patients. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy.