Beta class glutathione S-transferase (GST) activity is known to be associated with antibiotic resistance, one of the most serious threats to global health. In this research, the study of antibiotic resistance developed by beta class GST was conducted using KKSG6, one of the GST isozymes found in Acidovorax sp. KKS102. The KKSG6 gene has been successfully expressed in Escherichia coli BL21 Star™ (DE3) using pET101/D-TOPO®-KKSG6 as an expression vector, resulting in the presence of a protein band of approximatly 20 kDa after purification using GSTrap™ HP column. Over-expression of KKSG6 made Escherichia coli BL21 Star™ (DE3) to be less susceptible towards kanamycin, streptomycin, gentamycin, tetracycline and chloramphenicol, suggesting the antibiotics binding with KKSG6. Our study has revealed the KKSG6 conjugation activity towards chloramphenicol, but not with kanamycin and tetracycline. The inhibition of protein conjugation activity towards CDNB by the presence of chloramphenicol has also been demonstrated. An in-silico study using protein-ligand docking predicted that antibiotics binding could take place at the protein dimer interface and H-site depending on their properties.