CD45 is a leukocyte specific transmembrane glycoprotein and a receptor-like protein tyrosine phosphatase (PTP). CD45 can be expressed as several alternatively spliced isoforms that differ in the extracellular domain. The isoforms are regulated in a cell type and activation state-dependent manner, yet their function has remained elusive. The Src family kinase members Lck and Lyn are key substrates for CD45 in T and B lymphocytes, respectively. CD45 lowers the threshold of antigen receptor signalling, which impacts T and B cell activation and development. CD45 also regulates antigen triggered Fc receptor signalling in mast cells and Toll-like receptor (TLR) signalling in dendritic cells, thus broadening the role of CD45 to other recognition receptors involved in adaptive and innate immunity. In addition, CD45 can affect immune cell adhesion and migration and can modulate cytokine production and signalling. Here we review what is known about the substrate specificity and regulation of CD45 and summarise its effect on immune cell signalling pathways, from its established role in T and B antigen receptor signalling to its emerging role regulating innate immune cell recognition and cytokine production.