Protein phosphatase 5 (PP5) is a serine/threonine phosphatase that belongs to the PPP family phosphatases. PP5 and the other phosphatases of the PPP family share significantly similar catalytic domain structure. Due to this structural similarity, natural competitive inhibitors such as okadaic acid and cantharidin exhibit broad specificity over the PPP family phosphatases. In this study, we report the identification of three PP5 inhibitors, Ro 90-7501, aurothioglucose, and N-oleoyldopamine, along with a novel inhibitory mechanism of Ro 90-7501. Unlike other inhibitors binding to the phosphatase domain, Ro 90-7501 inhibited PP5 in a TPR-dependent manner. This TPR-dependent PP5 inhibition shown by Ro 90-7501 is a unique and novel inhibitory mechanism, which might be a useful tool for studies of PP5 on both regulatory mechanism and drug discovery. (C) 2016 Elsevier Inc. All rights reserved.