Legionella pneumophila infect eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and posttranslational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP) – a process referred to as AMPylation. Here, we used a chemical approach for stabilizing low affinity Rab:DrrA complexes in a site-specific manner to gain insights into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The X-ray crystal structure of the Rab:DrrA complex revealed a previously unrecognized non-conventional Rab binding site (NC-RBS). Biochemical characterizations demonstrated allosteric stimulation of DrrA’s AMPylation activity via Rab-binding to the NC-RBS. We propose that allosteric control of DrrA not only prevents random and potentially cytotoxic AMPylation in the host, thereby ensuring an efficient infection process by Legionella, but also represents an unprecedented AMPylation activation mechanism.