We attempted to establish the mesoscale layer-specific fMRI representation of neuronal activity using layer-specific Cre-driver mouse lines, optogenetic stimulations, fMRI and electrophysiological recordings. Although laminar fMRI responses were distinct during L2/3, L4, L5 and L6 stimulations, all fMRI responses increased along the cortical depth. This phenomenon was, however, not observed in LFP and spike recordings. This discrepancy between fMRI, LFP and spiking may be due to the draining veins transporting deoxyhemoglobin from the deeper layers to the superficial layers. Future studies may take into account of neurovasculature to elucidate the exact mechanisms of mesoscale layer-specific neurovascular coupling.