Most breast cancer deaths are not due to the growth of the primary tumour but rather to metastasis and the resulting impairment of function in the affected organ. Breast cancer metastasis to the brain is an increasing problem. In patients with advanced breast cancer, up to 30% have brain involvement. We have isolated a variant (4T1Br4) from the 4T1 syngeneic mouse model of triple negative breast cancer that metastasises preferentially to the brain, in up to 80% of mice. Affymetrix gene array profiling identified the serine/threonine survival kinase Pim-1 to be upregulated in brain metastatic tumours compared to tumours derived from parental or other related highly metastatic variants of the 4T1 model. Here, we confirmed the high expression of Pim-1 protein in mouse and xenograft models of breast cancer metastasis to brain by immunohistochemistry and show that Pim-1 mRNA expression correlates with the brain metastatic propensity of mouse and human breast cancer cell lines. Furthermore, we demonstrate that high expression of Pim-1 in brain-metastatic cells is associated with increased resistance to conventional chemotherapy as well as increased P-glycoprotein (Pgp) efflux pump activity. Thus, we hypothesised that Pim-1 may regulate chemo-resistance via the phosphorylation and/or activation of Pgp, resulting in increased efflux and therefore decreased retention of chemotherapeutic drug in brain-metastatic cells. Consistent with this, pharmacological inhibition of Pim-1 significantly increased the sensitivity of 4T1Br4 cells to chemotherapy and inhibited the phosphorylation and membrane expression of Pgp. We propose that Pim-1 may be a useful prognostic factor and therapeutic target for the treatment of brain-metastatic breast cancer.