Objectives To realize a practical technology for recycling both cyclodextrin and resting-cells at the same time in phytosterol biotransformation using mycobacterial resting cells. Results In order to produce 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) efficiently and low-costly, a recycled phytosterols (PS) biotransformation process using mycobacterial resting cells was developed. By optimizing the ratio of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and PS to 1:1 (w/w), most products crystallized during the biotransformation process. So, the HBC was easily separated by low-speed (900xg) centrifugation with yield of 92%. The resting cells, HP-beta-CD and the residual products and substrates left in the reaction system were reused for another bioconversion cycle after PS supplement. Three continuous cycles were achieved without the supplement of cells and HP-beta-CD. In each batch, 80 g L-1 of PS was transformed to HBC with the space-time yield of HBC of 8.9-12.8 g L(-1)day(-1). Conclusions This strategy reduced the cost of HBC production and simplified the purification process.