BACKGROUND AND PURPOSE: The development of antinociceptive morphine tolerance is a clinically intractable problem. Previous studies clarified the pivotal roles of platelet-derived growth factor (PDGF) and its receptor PDGFRβ in morphine tolerance. Herein, we investigated the role of spinal heat shock protein 27 (HSP27) in morphine tolerance and its relationship with PDGFRβ activation. EXPERIMENTAL APPROACH: Rats were treated with morphine for nine days and the effect of drug was evaluated by a tail-flick latency test. Western blot, real-time PCR, immunofluorescent staining, and various antagonists, agonists, and small interfering RNA lentiviral vectors elucidated the roles of HSP27, PDGFRβ, and related signaling pathways in morphine tolerance. KEY RESULTS: Chronic morphine administration significantly increased spinal cord HSP27 expression and phosphorylation. Downregulating HSP27 attenuated the development of morphine tolerance. PDGFRβ antagonism inhibited HSP27 activation, attenuated and reversed morphine tolerance. PDGFRβ induction increased HSP27 expression and activation and partially decreased morphine analgesia. PDGFRβ inhibition reduced Akt and p38 MAPK activity in morphine tolerance. PI3K and p38 inhibitors reversed morphine tolerance and suppressed morphine-induced HSP27 phosphorylation. CONCLUSION AND IMPLICATIONS: This study demonstrated for the first time that spinal HSP27 participates in PDGFRβ-mediated morphine tolerance via the PI3K/Akt and p38 MAPK signaling pathways. These findings implicated a potential clinical strategy for prolonging the opioid antinociceptive effect during long-term pain control. This article is protected by copyright. All rights reserved.