<p>The dorsal subiculum (dSub) and medial mammillary body (MM) are essential components of the extended hippocampal system, heavily implicated in spatial memory processes. However, the exact function of the prominent dSub-to-MM projection has remained ambiguous. Here, integrating c-fos mapping, fiber photometry, optogenetic manipulations and behavioral paradigms, we found that the monosynaptic connection between dSub and MM, rather than collaterals to other regions, is not only involved in spatial memory retrieval but also highly vulnerable to perturbations on activity level in both directions. In addition, we mapped the single neuron projectome of MM-projecting dSub neurons using fluorescence micro-optical sectioning tomography (fMOST). Our study unveiled that the individual dSub neuron extend efferent connections to the bilateral MM neurons, while also exhibiting substantial collateral projections to the retrosplenial cortex (RSP) and entorhinal (ENT). Our findings shed light on previously unknown subtypes and organizational principles of projection neurons in the afferent circuitry of the MM, thereby elucidating the circuit mechanism underlying the contribution of the dSub-MM circuit to spatial memory.</p>