Perioperative hypersensitivity (POH) to the neuromuscular blocking drug (NMBD) rocuronium was previously thought to be IgE and mast cell (MC)-mediated. However, the recent seminalobservation that rocuronium induces degranulation in murine peritoneal MCs (PMCs) via Mas-related G protein-coupled receptor B2 (MrgprB2) led to the idea that POH to this drug involves the activation of MRGPRX2 (human ortholog of MrgprB2). Furthermore, based on the demonstration that a patient with POH to rocuronium displayed three missense mutations (M196I, L226P and L237P) in MRGPRX2’s transmembrane domains, it was proposed that this hypersensitivity reaction resulted from aberrant activation of this receptor. We found that rocuronium at 20 μg/mL caused degranulation in mouse PMCs via MrgprB2 but required at least 500 μg/mL to induce degranulation in human MCs via MRGPRX2. Furthermore, RBL-2H3 cells transiently expressing M196I, L226P and L237P variants did not display enhanced degranulation in response to rocuronium when compared to the wild-type receptor. These findings provide the first demonstration that rocuronium induces degranulation in human MCs via MRGPRX2. Furthermore, the important differences between Mrg-prB2 and MRGPRX2 and the inability of rocuronium to induce enhanced response in cells expressing MRGPRX2 variants suggest that the mechanism of its POH is more complex than previously thought.