The medial entorhinal cortex (MEC) spatially modulated neurons, including grid cells, all contribute to the brain's spatial navigation system. The mechanisms behind the grid cells' hexagonal firing pattern in space remain elusive. One question pertains to how distal inputs modulate the grid cell network. The medial septum (MS) densely innervates the MEC and pharmacological inactivation of the MS is found to disrupt the spatial periodicity of grid cell firing and the local field potential (LFP) theta oscillations. The spiking activity of grid cells is in synchrony with the ongoing LFP theta oscillations, suggesting a relationship. In contrast, this relation is not found in bats that seem to lack theta activity, indicating that grid cell activity may be independent of theta oscillations. The MS projection to the MEC consists of inhibitory and excitatory neurons but their relative contributions are not well understood. Moreover, the postsynaptic targets of the MS projections in the MEC have not been thoroughly mapped out. The aim of the present study was to investigate the role of the MS projection in the grid cell network by anatomical and functional assessment of the connectivity of MS projections onto neurons in the MEC. To investigate the contribution of inhibitory and excitatory MS terminals in the MEC, we expressed a fluorescent tag together with the presynaptic component synaptophysin in rat MS neurons by use of a viral vector. Neurochemical mapping was conducted on brain sections for the characterisation of pre-and postsynaptic cells in the MEC. Co-localisation analyses were conducted from confocal images obtained from MEC layer II (LII) and III (LIII). To investigate how perturbations of the MS projection affected single unit and LFP activity in the MEC, we performed optogenetic perturbations of the MS projection and simultaneous electrophysiological recordings in the MEC. The preliminary neuroanatomical findings in the current study suggest that about 80% of MS terminals in the MEC are GABAergic. The postsynaptic targets of the projection are both calbindin positive pyramidal LII cells and parvalbumin positive LII and LIII cells. The parvalbumin positive cells in LII received nearly double the input compared to the other two cell populations. The optogenetic perturbations of the MS projections indicated that the grid cells' firing rate increased with increased power in the LFP theta with no change in the spatial modulation. Activating the MS-MEC projection caused an instantaneous inhibition of narrow spiking putative interneurons, whereas grid cells showed a delayed increase in response to activation. The anatomical findings suggest direct inhibition of the MS projection onto PV positive interneurons most prominently in MEC LII. Preliminary data from the electrophysiology suggest that the spatial coding of grid cells may be dissociated from LFP theta oscillations, and that the MS may act in disinhibition of the grid cells, through long-range inhibitory neurons.