A heterotrophic bacterium, designated as HIB4, having an ability to oxidize ferrous iron was isolated from the sample of an enrichment culture with 9K medium, by using the modified WAYE (washed agarose/yeast extract) medium with ferrous sulphate. This isolate was identified as Alicyclobacillus disulfidooxidans from 16S rDNA sequence analysis. The isolate grew and oxidized ferrous iron in an inorganic medium containing 0.02 % (w/v) of yeast extract and Ferrous iron oxidation occurred at the almost end of its logarithmic phase. Yeast extract was an essential substrate for the isolate because the isolate could not grow or oxidize ferrous iron without yeast extract. However, higher concentration of yeast extract inhibited the growth of the isolate. On the other hand, it was confirmed that the isolate was able to grow without ferrous ion so that it did not get any energy by ferrous ion oxidation. Its optimum concentration of yeast extract was 0.02% (w/v) at the concentration of ferrous ion 0.08mol/liter. Its optimum pH was 1.5 and the optimum temperature was 30 °C These physiological characteristics were completely different from A. disulfidooxidans SD-11 which is the type strain.