Plazomicin, a next-generation aminoglycoside, was recently approved by the U.S. Food and Drug Administration for the treatment of multidrug-resistant complicated urinary tract infections. It is highly effective against most aminoglycoside-modifying enzymes (AMEs). Here, we report that Salmonella enterica strains containing the newly identified gentamicin resistance gene (grdA) are highly resistant to plazomicin. Heterologous expression of grdA in Escherichia coli ΔtolC resulted in plazomicin resistance with minimum inhibitory concentration (MIC) > 256 µg/mL. These findings reveal that GrdA confers significantly higher resistance to plazomicin than the previously known plazomicin-resistant AMEs AA (2)-Ia and APH (2″)-Iva.