RAC1 signalling has been implicated in a variety of dynamic cell biological processes that are orchestrated through regulated localisation and activation of RAC1. As a small GTPase, RAC1 switches between active and inactive states at various subcellular locations that include the plasma membrane, nucleus and mitochondria. Once activated, RAC1 interacts with a range of effectors that then mediate various biological functions. RAC1 is regulated by a large number of proteins that can promote its recruitment, activation, deactivation, or stability. RAC1 and its regulators are subject to various post-translational modifications that further fine tune RAC1 localisation, levels and activity. Developments in technologies have enabled the accurate detection of activated RAC1 during processes such as cell migration, invasion and DNA damage. Here, we highlight recent advances in our understanding of RAC1 regulation and function at specific subcellular sites.