Arsenite-tolerant bacteria were isolated from an organic farm of Navsari Agricultural University (NAU), Gujarat, India (Latitude: 20°55′39.04″N; Longitude: 72°54′6.34″E). One of the isolates, NAU-1 (aerobic, Gram-positive, non-motile, coccobacilli), was hyper-tolerant to arsenite (AsIII, 23 mM) and arsenate (AsV, 180 mM). 16S rRNA gene of NAU-1 was 99% similar to the 16S rRNA genes of Rhodococcus (Accession No. HQ659188). Assays confirmed the presence of membrane bound arsenite oxidase and cytoplasmic arsenate reductase in NAU-1. Genes for arsenite transporters (arsB and ACR3(1)) and arsenite oxidase gene (aoxB) were confirmed by PCR. Arsenite oxidation and arsenite efflux genes help the bacteria to tolerate arsenite. Specific activities of antioxidant enzymes (catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase) increased in dose-dependent manner with arsenite, whereas glutathione reductase activity decreased with increase in AsIII concentration. Metabolic studies revealed that Rhodococcus NAU-1 produces excess of gluconic and succinic acids, and also activities of glucose dehydrogenase, phosphoenol pyruvate carboxylase and isocitrate lyase were increased, to cope with the inhibited activities of glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase enzymes respectively, in the presence of AsIII. Enzyme assays revealed the increase in direct oxidative and glyoxylate pathway in Rhodococcus NAU-1 in the presence of AsIII.