In order to better understand the role of histamine H4 (H4R) receptor in breast cancer, we studied the receptor expression pattern, associated signal transduction pathway and biological responses, in breast cancer cell lines with different malignant characteristics. A different pattern of protein expression was observed in MDA-MB-231 compared to MCF-7 cells determined by western blot, exhibiting the presence of a diverse range of molecular weight species of the H4R. H4R agonist reduced cyclic adenosine monophosphate (cAMP) formation induced by forskolin only in MCF-7 cells. In MDA-MB-231 cells, H4R agonists significantly decreased cell proliferation, augmented the Annexin-V and TdT-mediated UTP-biotin Nick End labelling (TUNEL) positive cells and produced a 2.5-fold increase in cell senescence. In MCF-7 cells, H4R agonists inhibited proliferation by 50%, increasing the exponential doubling time. This effect was associated to an augment in Annexin-V and TUNEL positive cells, and a 2-fold increase in cell senescence. We conclude that H4R is functionally expressed in human breast cancer cell lines, exhibiting a key role in histamine-mediated biological processes such as cell proliferation, senescence and apoptosis.