Dexmedetomidine (DEX) a type of the anaesthetic that has been widely used in anaesthesia and intensive care. However, whether DEX affects the pharmacokinetics of drugs remains elusive. As hepatic P-glycoprotein (P-gp) serves a critical role in the disposition of drugs, the present study aimed to address whether P-gp function could be affected by DEX in vitro. In the present study, L02 cells (a normal human liver cell line) were exposed to DEX for 24 h and P-gp function was evaluated by the intracellular accumulation of Rhodamine 123. The results indicated that P-gp function was significantly impaired by DEX treatment and that the mRNA levels and protein levels of P-gp were downregulated in a dose- and time-dependent manner. Importantly, DEX-induced downregulation of P-gp was associated with adenosine 5′-monophosphate-activated protein kinase (AMPK) activation, as it was significantly attenuated by AMPK inhibition using dorsomorphin. Furthermore, the results revealed that changes in the subcellular localisation of nuclear factor (NF)-κB following AMPK activation were involved in the P-gp regulation in response to DEX treatment. Collectively, these results suggested that DEX impairs P-glycoprotein-mediated efflux function in L02 cells via the AMPK/NF-κB pathway, which provided direct evidence that the hepatic disposition of drugs may be affected by DEX through the downregulation of P-gp.