BackgroundPaenibacillus thiaminolyticus, a species of genusPaenibacillusof the familyPaenibacillaceae, exists widely in environments and habitats in various plants and worms, and occasionally causes human infections. This work aimed to characterize the function of a novel aminoglycosideO-nucleotidyltransferase resistance gene, designatedant(6)-If, from aP. thiaminolyticusstrain PATH554.MethodsMolecular cloning, antimicrobial susceptibility testing, enzyme expression and purification, and kinetic analysis were used to validate the function of the novel gene. Whole-genome sequencing and comparative genomic analysis were performed to investigate the phylogenetic relationship of ANT(6)-If and other aminoglycosideO-nucleotidyltransferases, and the synteny ofant(6)-Ifrelated sequences.ResultsThe recombinant with the clonedant(6)-Ifgene (pMD19-ant(6)-If/DH5α) demonstrated a 128-fold increase of minimum inhibitory concentration level against streptomycin, compared with the control strains (DH5α and pMD19/DH5α). The kinetic parameterkcat/Kmof ANT(6)-If for streptomycin was 9.01 × 103M−1·s−1. Among the function-characterized resistance genes, ANT(6)-If shared the highest amino acid sequence identity of 75.35% with AadK. Theant(6)-Ifgene was located within a relatively conserved genomic region in the chromosome.Conclusionant(6)-Ifconferred resistance to streptomycin. The study of a novel resistance gene in an unusual environmental bacterium in this work contributed to elucidating the resistance mechanisms in the microorganisms.