Background: Treatment for estrogen receptor-positive (ER+)/HER2-negative advanced breast cancer (ABC) has undergone a revolution with the approval of cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). Biomarkers of treatment resistance are limited, though growing evidence points towards antitumor immune response as a key factor. We aimed to investigate immune-mediated resistance to CDK4/6i. Methods: Gene expression (GE) was analyzed in 63 FFPE tumors from 55 patients with ER+/HER2- BC (34 primary, 21 metastatic (M1) and 8 paired biopsies) treated with CDK4/6i. Breast Cancer 360 panel (nCounter NanoString Technologies) including intrinsic subtyping (IS). Patients were stratified into good/poor response (GR/PR), based on data from pivotal studies considering both hormone sensitivity and progression-free survival (PFS): 66,5% were GR and 37,5% PR. Multiple T-testing was used to assess GE differences and Kaplan Meier methods and Cox regression models for survival. Results: Thirty-six patients (65.5%) received CDK4/6i in first line. 16 deaths were notified at the time of the analysis. Most tumors were luminal A or B (n = 23;36.5%), 11.1% HER2-enriched (n = 7) and 3.2% Basal-like (n = 2). 54.9% patients had visceral M1 before CDK4/6i. In first line, patients with luminal A, followed by luminal B tumours showed longer PFS compared to HER2-enriched (p = 0.02). The presence of visceral M1 and PR to CDK4/6i were associated with significantly shorter PFS and overall survival (OS) (p < 0.05), remaining as independent predictors after adjustment by the standard clinico-pathological variables. To investigate other factors driving resistance to CDK4/6i, GE differences between response groups was compared. Increased GE of several immune-related features was associated with PR using the M1 samples, including macrophages, PDL2, IFN-gamma and inflammatory-chemokines; (log2C (-0.9)-(-1.2); p-value < 0.05); while only few characteristics were significative using primary biopsies (Homologous recombination deficiency, log2FC -1.01, p = 0.02; Likelihood test for model comparison: p-value < 0.001). High GE of macrophages signature was associated with worse OS (HR: 3.14; CI 1.3-7.6, p = 0.03), and progesterone receptor with better OS (HR:0.78; CI95%0.64-0.95, p = 0.04). Other genes related to immunity/inflammation (CD68, CD163, CCR1 or CDC25B), were also associated with worse OS (HR 1.8-6.1; p-value < 0.05), further pointing towards immune response as a key determinant of CDK4/6i efficacy. Conclusions: Our study suggests a key role of tumor immunity in resistance to CDK4/6i, with an additional association of macrophages with worse outcome. The correlation of poor response to CDK4/6i with worse survival suggests its utility as a surrogate biomarker. Further investigation of the use of immunotherapeutic agents in this setting is deserved to improve CDK4/6i response.