The aminoglycoside phosphotransferase (APH(3')-IIIa) kinases form a clinically central group of antibiotic-resistant enzymes. Computationally, we have studied the catalytic mechanism of the APH(3')-IIIa enzyme at the atomic-level. The proposed reaction mechanism involves protonation of Asp190 by the kanamycin 3'-hydroxyl group mediated through an explicit neighboring water molecule, which leads to a simultaneous nucleophilic attack on the gamma-phosphate of the ATP by the deprotonated kanamycin 3'-hydroxyl group. The second step is a proton abstraction from the protonated Asp190 to the phosphate group of the phosphorylated kanamycin mediated by an explicit water molecule. The calculated Gibbs energy of activation (Delta G(double dagger)) of the rate-determining step for the phosphorylation reaction is 77 kJ mol(-1 )at the M06-2X/6-311++G(2df,p)//ONIOM(M06-2X/6-31+G(d):HF/6-31G(d)) level of theory. This study has provided a new understanding of the APH(3')-IIIa catalytic mechanism that agrees with the available experimental data (Delta G(double dagger) = 75 +/- 4 kJ mol(-1)) and could provide a starting point for the rational design of mechanism-based inhibitors of aminoglycoside modifying enzyme to circumvent antibiotic resistance.