The function, trafficking and synaptic signalling of AMPA receptors are tightly regulated by phosphorylation. CaMKII phosphorylates the GluA1 AMPA subunit at Ser831 to increase single channel conductance. We show for the first time that CaMKII increases the conductance of native heteromeric AMPA receptors in mouse hippocampal neurons via phosphorylation of Ser831. In addition, co-expression of TARPs with recombinant receptors is required for phosphoSer831 to increase conductance of heteromeric GluA1/GluA2 receptors. Finally, phosphorylation of Ser831 increases the efficiency with which each subunit can activate, independent of agonist efficacy, thereby increasing the likelihood that more receptor subunits will be simultaneously activated during gating. This underlies the observation that phosphoSer831 increases the frequency of openings to larger conductance levels rather than altering unitary conductance. Together, these findings suggest that CaMKII phosphorylation of GluA1-Ser831 decreases the activation energy for an intrasubunit conformational change that regulates the conductance of the receptor when the channel pore opens.