Updated project metadata. Upon cellular stress, the concentration of the alarmone diadenosine triphosphate (Ap3A) increases and is thought to trigger downstream adaptive processes. The known Ap3A-hydrolase fragile histidine triad (Fhit) is known to form a complex with Ap3A. The molecular mechanism of Fhit-Ap3A signaling is however unknown. In this study, by synthesizing a covalent Fhit-Ap3A complex we have elucidated the Ap3A-dependent cellular interactome by mass spectrometry-based proteome profiling. Interestingly, we found a significant enrichment of proteins involved in translation. We then show that Fhit translocates from the nucleolus into the cytosol upon stress to form a Fhit-Ap3A complex which impedes translation both in vitro and in vivo in an Ap3A dependent manner resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap3A to regulate cellular proliferation.