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β-cyclodextrins derivatives (CDs) have applied in steroids biotransformation industry because of their unique properties. However, the effect of β-CDs on the growth rate, activity, conversion, and characters of whole cells has not been concerned. In this study, the growth rate and cellular morphology of Arthrobacter simplex (ASP) pretreated by six kinds of β-CDs were measured. The results showed that most β-CDs inhibited the growth of ASP, among which randomly methylated-β-CDs has the most serve inhibition; however, sulfonic acid-β-CD promoted the growth of cells. The morphology size and the surface of all β-CDs-pretreated cells were changed compared with the control group. Besides, the conversion of cortisone acetate (CA) increased in β-CDs-pretreatment system and β-CDs-containing system, which reached 97.98% in hydroxypropyl-β-cyclodextrin (HP-β-CD) containing-system and 78.69% in HP-β-CD-pretreatment-system, but the dehydrogenase activity of all β-CDs-pretreated cells decreased. β-CDs with higher K value have stronger inclusion ability with CA, and along with the membrane permeability of β-CDs-pretreated cells increased more, but they also have more serve damage on the ASP cells, which is negative to increase the conversion of CA. This study improved our understanding of the effect of β-CDs when they were used in the steroids biotransformation by ASP whole cells, and provided data basis for the selection of suitable CDs for application.