Detoxification of Cr(VI) under alkaline pH requires attention due to the alkaline nature of many effluents. An alkaliphilic gram-positive Bacillus subtilis isolated from tannery effluent contaminated soil was found to grow and reduce Cr(VI) up to 100% at an alkaline pH 9. Decrease in pH to acidic range with growth of the bacterium signified the role played by metabolites (organic acids) in chromium resistance and reduction mechanism. The XPS and FT-IR spectra confirmed the reduction of Cr(VI) by bacteria into +3 oxidation state. Chromate reductase assay indicated that the reduction was mediated by constitutive membrane bound enzymes. The kinetics of Cr(VI) reduction activity derived using the monod equation proved (K s = 0.00032) high affinity of the organism to the metal. This study thus helped to localize the reduction activity at subcellular level in a chromium resistant alkaliphilic Bacillus sp.