Activity patterns of individual cortical neurons change during brain-computer interface performance and motor adaptation. To investigate to what extent the modulation of individual neurons is affected by local ensemble interactions and global modulatory signals, we conducted two-photon calcium imaging of layer 2/3 neurons in the mouse motor cortex and simultaneously reinforced the activity of a single neuron with water delivery. The reinforcement increased the activity of the target neuron within 15 min and bi-directionally modulated the activity of a subset of non-target neurons. This bidirectional modulation depended on the relative timing between reward delivery and the neuronal activity, but not on the functional correlation with or distance from the target neuron. The bidirectional modulation was recreated by repetitive pairing of reward delivery and photoactivation of cortical neurons. The reward-timing-dependent bidirectional modulation may be a fundamental process in the layer 2/3 microcircuit reorganization for fast adaptation to novel environments.