Breast cancer (BC) cells exploit the up-regulation or down-regulation of immune checkpoint molecules (ICM) to evade anti-tumor immune responses.ICMs can be measured in human plasma; however, their biological and clinical significance remains essentially unknown. The present analysis aimed to measure plasma ICMs in metastatic BC patients (pts) and compare them to healthy controls. Soluble forms of ICM and RANTES, arginase, TGF-b1, CD163, and CD206 were measured using Multiplex® bead array and ELISA technologies. Plasma samples from 20 metastatic breast cancer (MBC) pts and 45 healthy controls were analyzed for each protein, and compared between MBC pts and healthy controls using a non-parametric test (Mann-Whitney). The median age of the cohort was 53 years (range 34-79 years). The performance status was as follows; PS=0 (11 pts), PS=1 (7 pts) and PS=2 (2 pts). The soluble co-stimulatory molecules, GITR (p<0.0011), GITRL (p< 0.0000), CD27 (p< 0.0039), CD28 (p<0.0069), CD40 (p< 0.0022), CD86 (p< 0.0000) and ICOS (p< 0.0157), as well as the co-inhibitory molecules, PD-L1 (p< 0.0002), CTLA-4 (p< 0.0002) and BTLA (p<0.0145) levels were significantly lower in MBC pts compared to healthy controls. Conversely, the co-inhibitory molecules, TIM-3 (p< 0.0001) and LAG-3 (p<0.0000 were significantly higher than those of healthy controls. Other biomarkers with raised plasma concentrations included TLR (p<0.0039). Plasma CD80 (p<0.0992), PD-1 (p<0.2325), HVEM (p<0.062,6), RANTES (p<0.4861) CD163 (p<0,8565) and CD206 (p<0,2454) levels were not significantly different between the MBC pts and the healthy controls. We identified lower levels of CD27, CD28, CD40, ICOS, GITR, GITRL, CD86, PD-L1, CTLA-4, BTLA, arginase, and TGF-β1, and higher levels of TIM-3 and LAG-3 immune checkpoint molecules in MBC pts compared to healthy controls. These results indicate that a down-regulation of soluble ICM pathways and an up-regulation of some inhibitory ICM pathways are associated with MBC patients. To our knowledge, this is the first study to describe soluble immune checkpoint molecules in MBC pts.