Antimicrobial resistant pathogens are a global health threat driven by the indiscriminate use of antimicrobials. Antimicrobial resistance can be acquired by resistance genes encoded by mobile genetic elements. In this study, we identified a strain of Salmonella enterica serovar Gallinarum (SG4021) from an infected chicken in Korea and characterized the presence of resistance genes in its plasmid by whole genome sequencing. The sequence was then compared with that of a plasmid (P2) from strain SG_07Q015, the only other strain of S. Gallinarum isolated in Korea for which a genome sequence is available. The results revealed that both strains harbored nearly identical DNA carrying antibiotic resistance gene cassettes inserted into integron In2 of the transposable element Tn21, namely an aadA1 resistance gene conferring resistance to aminoglycosides and a sul1 resistance gene conferring resistance to sulfonamide. Interestingly, despite the presence of sul1 in SG4021, an antibiotic sensitivity test revealed that it was sensitive to sulfonamides. Further analysis revealed that this disparity was due to the insertion of a ~ 5 kb ISCR16 sequence downstream of the promoter driving sul1 expression in SG4021. Using various mutants, we showed that the insertion of ISCR16 blocked the expression of the sul1 gene from the upstream promoter. Therefore, the functionality of antimicrobial resistance genes determines phenotypic antimicrobial resistance.