Morphological remodeling of dendritic spines is critically involved in memory formation and depends on adhesion molecules. Serotonin receptors are also implicated in this remodeling, though the underlying mechanisms remain enigmatic. Here, we uncovered a signaling pathway involving the adhesion molecule L1CAM (L1) and serotonin receptor 5-HT4 (5-HT4R, encoded by HTR4). Using Förster resonance energy transfer (FRET) imaging, we demonstrated a physical interaction between 5-HT4R and L1, and found that 5-HT4R-L1 heterodimerization facilitates mitogen-activated protein kinase activation in a Gs-dependent manner. We also found that 5-HT4R-L1-mediated signaling is involved in G13-dependent modulation of cofilin-1 activity. In hippocampal neurons in vitro, the 5-HT4R-L1 pathway triggers maturation of dendritic spines. Thus, the 5-HT4R-L1 signaling module represents a previously unknown molecular pathway regulating synaptic remodeling.