Aims:The aim of this study was to evaluate the diversity and potential for horizontal transfer of amikacin resistance genes from the human gut. Methods and results:A library of human faecal microbiota was constructed and subjected to functional screening for amikacin resistance. In total, five amikacin resistance genes that conferred relatively high amikacin resistance, with minimum inhibitory concentrations (MICs) ranging from 64 to >512, were identified from the library, including a novel aminoglycoside acetyltransferase gene and a 16S rRNA methyltransferase (MTase) gene, labelled aac (6′)-Iao and rmtI, respectively. AAC(6′)-Iao showed the highest identity of 48% to AAC(6′)-Ian from a clinical isolate Serratia marcescens, whereas RmtI shared the closest amino acid identity of 32% with ArmA from Klebsiella pneumonia. The MICs of these five subclones to six commonly used aminoglycosides were determined. Susceptibility analysis indicated that RmtI was associated with high resistance phenotype to 4,6-disubstituted 2-DOS aminoglycosides, whereas AAC(6′)-Iao conferred resistance to amikacin and kanamycin. In addition, kinetic parameters of AAC(6′)-Iao were determined, suggesting a strong catalytic effect on amikacin and kanamycin.Conclusions: Antibiotic resistance genes with low identity to known sequences can be uncovered by functional metagenomics. In addition, the diversity and prevalence of amikacin resistance genes merit further investigation in extended habitats, especially the 16S rRNA MTase gene that might have been underestimated in previous cognition.Significance and Impact of Study: Two novel amikacin resistance genes were identified in this study, including a 16S rRNA methyltransferase gene rmtI and an aminoglycoside acetyltransferase gene aac(6′)-Iao. This work would contribute to the in-depth study of the diversity and horizontal transfer potential of amikacin resistance genes in the microbiome of the human gut.