<p>Understanding the antibiotic resistance profile of the emerging pathogen <i>Citrobacter telavivum</i> S24 is important for clinical care, surveillance, and research. <i>C. telavivum</i> S24, a strain that exhibits resistance to both streptomycin and spectinomycin, was isolated from the soil at a broiler chicken farm. The strain was identified by whole-genome sequencing and average nucleotide identity analyses. Annotation of the resistance genes revealed that a novel aminoglycoside resistance gene, designated <i>ant(3″)-Ic</i>, is located on the chromosome of <i>C. telavivum</i> S24. This gene exhibits 52% similarity with the previously functionally characterized resistance gene <i>aadA23</i>. It distinguishes itself from <i>ant(3″)-Ia</i> and <i>ant(3″)-Ib</i> genes, and represents an independent branch. The <i>ant(3″)-Ic</i> gene is recognized as an inherent resistance gene of <i>C. telavivum</i> within a conserved genetic environment. The product of the <i>ant(3″)-Ic</i> gene is the aminoglycoside nucleotidyltransferase ANT(3″)-Ic, which has a theoretical pI of 5.05 and a molecular mass of 29&#xa0;kDa. ANT(3”)-Ic inactivates streptomycin and spectinomycin with the <i>K</i>_m of 46.13 and 115.90 µM, respectively. The discovery of the novel antibiotic resistance gene <i>ant(3″)-Ic</i> and the functional characterization of ANT(3”)-Ic will facilitate more targeted surveillance and treatment strategies for preventing and controlling zoonotic pathogens of zoonotic diseases.</p>