Fentanyl is a frequently used and abused opioid analgesic and can cause internalization of mu opioid receptors (MORs). Receptor internalization modulates the signaling pathways of opioid receptors. As changes in dendritic spines and synaptic AMPA receptors play important roles in addiction and memory loss, we investigated how fentanyl affects dendritic spines and synaptic AMPA receptors in cultured hippocampal neurons. Fentanyl at low concentrations (0.01 and 0.1 microM) caused the collapse of dendritic spines and decreased the number of AMPA receptor clusters. In contrast, fentanyl at high concentrations (1 and 10 microM) had opposite effects, inducing the emergence of new spines and increasing the number of AMPA receptor clusters. These dose-dependent bidirectional effects of fentanyl were blocked by a selective MOR antagonist CTOP at 5 microM. In neurons that had been transfected with HA-tagged or GFP-tagged MORs, fentanyl at high concentrations induced persistent and robust internalization of MORs, whereas fentanyl at lower concentrations induced little or transient receptor internalization. The blockade of receptor internalization with the expression of dominant-negative Dynamin I (the K44E mutant) reversed the effect of fentanyl at high concentrations, supporting a role of receptor internalization in modulating the dose-dependent effects of fentanyl. In contrast to morphine, the effects of fentanyl on dendritic spines are distinctively bidirectional and concentration dependent, probably due to its ability to induce robust internalization of MORs at high concentrations. The characterization of the effects of fentanyl on spines and AMPA receptors may help us understand the roles of MOR internalization in addiction and cognitive deficits.