Microglia serve important roles in chronic pain signal transduction pathways. Glia cells, especially microglia, seem to share mechanisms that lead to chronic pain and morphine‑induced tolerance. Evidence has suggested that downregulating cytoskeleton activity in microglia provides pain relief in chronic pain and morphine tolerance. The purpose of the present study was to evaluate the effect of ethanol extracts of Hericiumerinaceus (EHE) mycelium on morphine‑induced BV2 microglial cell activation. BV2 cells were starved for 4h in DMEM before being incubated with 100ng/ml EHE for 30 min, followed by 1µM morphine for 2h. Subsequently, the cells were harvested and used for migration experiments and western blotting. The results showed that 1µM morphine enhanced BV2 cell activation and chemotactic reaction, and it increased histone deacetylase 6 (HDAC6) expression and heat shock protein 90 (HSP90) deacetylation as well as HSP90 cleavage. Pretreatment with 100ng/ml EHE significantly inhibited the morphine‑stimulated effects on BV2 cells. The present study demonstrated that EHE inhibited morphine‑induced BV2 activations by regulating the HDAC6/HSP90 deacetylation signal transduction pathway.