Synapses established during central nervous system development can be modified through synapse elimination and formation. These processes are, in part, activity dependent and require regulated trafficking of post‐synaptic components. Here, we investigate the activity‐driven remodeling of cultured rat hippocampal neurons at 14 days in vitro, focusing on the post‐synaptic proteins PSD‐95, Shank, neuroligin (NL)1 and actin. Using live imaging and photoconductive stimulation, we found that high‐frequency activity altered the trajectory, but not velocity, of PSD‐95‐GFP and Shank‐YFP clusters, whereas it reduced the speed and increased the number of NL1 clusters. Actin‐CFP reorganized into puncta following activity and ∼50% of new puncta colocalized with NL1 clusters. Actin reorganization was enhanced by the overexpression of NL1 and decreased by the expression of an NL1 mutant, NL1‐R473C. These results demonstrate activity‐dependent changes that may result in the formation of new post‐synaptic sites and suggest that NL1 modulates actin reorganization. The results also suggest that a common mechanism underlies both the developmental and activity‐dependent remodeling of excitatory synapses.