<b><i>Objectives:</i></b> Determining the effect of membrane-impermeant thiol/disulfide exchange inhibitors on rhesus rotavirus infectivity in MA104 cells and investigating protein disulfide isomerase (PDI) as a potential target for these inhibitors. <b><i>Methods:</i></b> Cells were treated with DTNB [5,5-dithio-bis-(2-nitrobenzoic acid)], bacitracin or anti-PDI antibodies and then infected with virus. Triple-layered particles (TLPs) were also pretreated with inhibitors before inoculation. The effects of these inhibitors on α-sarcin co-entry, virus binding to cells and PDI-TLP interaction were also examined. FACS analysis, cell-surface protein biotin-labeling, lipid-raft isolation and ELISA were performed to determine cell-surface PDI expression. <b><i>Results:</i></b> Infectivity became reduced by 50% when cells or TLPs were treated with 1 or 6 m<i>M</i> DTNB, respectively; infectivity became reduced by 50% by 20 m<i>M</i> bacitracin treatment of cells whereas TLPs were insensitive to bacitracin treatment; anti-PDI antibodies decreased viral infectivity by about 45%. The presence of DTNB (2.5 m<i>M</i>) or bacitracin (20 m<i>M</i>) was unable to prevent virus binding to cells and rotavirus-induced α-sarcin co-entry. <b><i>Conclusions:</i></b> It was concluded that thiol/disulfide exchange was involved in rotavirus entry process and that cell-surface PDI was at least a potential target for DTNB and bacitracin-induced infectivity inhibition.