Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.