ABSTRACT Research over the past two decades has highlighted that mechanical signaling is a crucial component in regulating biological processes. Although many processes and proteins are termed ‘mechanosensitive’, the underlying mechanisms involved in mechanosensing can vary greatly. Recent studies have also identified mechanosensing behaviors that can be regulated independently of applied force. This important finding has major implications for our understanding of downstream mechanotransduction, the process by which mechanical signals are converted into biochemical signals, as it offers another layer of biochemical regulatory control for these crucial signaling pathways. In this Review, we discuss the different molecular and cellular mechanisms of mechanosensing, how these processes are regulated and their effects on downstream mechanotransduction. Together, these discussions provide an important perspective on how cells and tissues control the ways in which they sense and interpret mechanical signals.