Proper development of neurons in the hippocampus is essential for learning and memory. Our laboratory previously discovered a family of synaptic adhesion-like molecules (SALMs) which induce neurite outgrowth in this brain region (Wang et al., 2006). Here we establish flotillin-1 (flot-1) as a molecular mediator of neurite branching for SALM4. Knockdown of flot-1 alone in cultured hippocampal neurons using siRNA from 3-7days in vitro (DIV) impaired neurite branching, whereas overexpression of flot-1 during the same time period increased the number of processes and branching. We show that induction of neurite outgrowth by flot-1 depends on amino acids 134-151 as well as lipid raft microdomains, SoHo proteins to regulate the actin cytoskeleton, and the exocyst complex to deliver new membrane proteins to growing neurites. When each of SALMs 1-5 was overexpressed, siRNA knockdown of flot-1 prevented neurite branching by SALM4. Overall, our data reveal a flot-1 signaling pathway for hippocampal neurite branching that is regulated by SALM4.