Background: Despite potential clinical implications, the complexity of immune microenvironment in breast cancer (BC) brain metastases (BM) is still poorly understood. Multiplex immunofluorescence (mIF) allows simultaneous visualization of several IF labeled proteins while maintaining spatial information. This novel technique can be used to comprehensively describe BCBM immune microenvironment, potentially providing useful information to guide novel therapeutic approaches. Methods: Clinical data and archival BM samples from 60 BC patients undergoing neurosurgery (2003-2018) at three institutions were collected. BCBMs were characterized using a custom mIF panel, including immune checkpoint and co-inhibitory molecules (CD3, PD1, PD-L1, TIM3, LAG3, CD163) and localization (keratin for tumor recognition) markers. Mean marker density was determined by digital image analysis (positive cells/mm2) and classified in tumor and stroma areas. Associations between immune marker densities, BC subtype and overall survival from BM diagnosis (OS) were studied. Results: Sixty BCBM samples were analyzed; 32% HR+/HER2-, 38% HER2+, 30% HR-/HER2-. At a median follow-up of 43 months, the only clinical variable associated with OS was BC subtype (shortest for HR-/HER2- and longest for HER2+, p=0.02). In the total sample area and tumor area, no significant difference in marker density was observed according to BC subtype. In the stroma area, a significant difference in TIM3+ cell density was observed according to BC subtype (highest density in HR+/HER2- and lowest density in HER2+ tumors, Kruskal-Wallis p=0.017). Higher CD163 density (a marker of M2 macrophage polarization), both in the tumor and in the stroma area, was significantly associated with worse OS, even after correction by BC subtype. In the subgroup of patients with HR+/HER2- BCBM, high TIM3+ cell density in the stroma area was significantly associated with longer OS (median OS 54.1 versus 23 months respectively for TIM3+ density above and below median value; p=0.01). Conclusions: In BCBM, stromal TIM3+ immune infiltrate differs according to BC subtype. M2 macrophage polarization is consistently associated with worse OS across all BC subtypes and might represent a potential therapeutic target for these patients.