The anterior cingulate cortex (ACC) serves as a critical hub for the anxiety and pain perception. The large-conductance Ca-activated potassium channels, or BK channels, are ubiquitously expressed throughout the central nervous system including the cingulate cortex. However, what changes of cortical BK channels undergo in the ACC remains unknown in pain-related anxiety. In the present study, a significant upregulation of synaptic and non-synaptic BK channel accessory β4 subunits in the ACC was accompanied with pain-associated anxiety-like behaviors in the chronic compression of multiple dorsal root ganglia (mCCD) of the rat. NS1619, an opener of BK channels, significantly rescued the alteration of fAHP and AP duration of ACC pyramidal neurons in mCCD rats. The mRNA expression of BK β4 subunits was extremely upregulated in the ACC after mCCD with the increased amount of both synaptic and non-synaptic BK β4 subunit protein. Meanwhile, NS1619 reversed the enhanced AMPA receptor-mediated spontaneous excitatory postsynaptic current (sEPSC) frequency and the attenuated PPR of ACC neurons in mCCD rats. Local activation of BK channels in the ACC reversed mechanical allodynia and anxiety-like behaviors. These results suggest that the upregulation of postsynaptic and presynaptic BK β4 subunit may contribute to neuronal hyperexcitability and the enhanced synaptic transmission in the ACC in neuropathic pain state, and then may result in anxiety-like behavior induced by neuropathic pain.