NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance. Display omitted •The first room-temperature structure of NendoU from SARS-CoV-2 is solved at an XFEL•Designed RNA substrates are cleaved within microcrystals of NendoU•High B factors indicate varying flexibility between trimers of the NendoU hexamer•Alternating trimer flexibility suggests a binding-change mechanism NendoU from SARS-CoV-2 is a protein responsible for the virus’s ability to evade the immune system. Jernigan et al. report the room-temperature structure of NendoU, finding indications that it functions by an alternate-switch mechanism. This sets a benchmark for studies targeting this enzyme in treating coronavirus infections.