Microglia-mediated synaptic pruning is an essential process in normal postnatal brain development. In certain regions of the brain, microglia have been shown to refine neuronal synaptic connections through the complement system. Based on structural similarities to complement regulatory proteins, as well as inhibitory effects on the complement system demonstrated in vitro, neuronal Seizure-related 6 (Sez6) family proteins are proposed to regulate microglial engulfment of synapses. Furthermore, Sez6 family proteins are prominent BACE1 substrates and therapeutic BACE inhibition (BACEi) is predicted to interfere with neuron-microglia signalling. In this study, the effects of the lack of Sez6 proteins on synaptosome and microglial properties were investigated. Quantitative proteomics of synaptosomes prepared from brains of mice lacking the three members of the Sez6 family (Sez6 triple knockout, or TKO), revealed significant decreases in signature microglial proteins including complement receptor 3 (CR3). To determine the functional effect of the lack of Sez6 family proteins on microglial pruning, live microglial engulfment assays were conducted. Microglia were found to preferentially engulf synaptosomes of the same genotype as their own when given a choice, although wild-type (WT) microglia engulfed significantly more synaptosomes than Sez6 TKO microglia, and WT synaptosomes were engulfed more than TKO synaptosomes by microglia of either genotype. Preliminary results from chemotaxis assays indicated that synaptosomes containing Sez6 proteins enhance the directional motility of WT, but not Sez6 TKO microglia. Taken together, these in vitro experimental results confirm that Sez6 proteins regulate microglial motility and synaptic engulfment.