The activity-dependent regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and the stabilization of synapses are critical to synaptic development and plasticity. One candidate molecule implicated in maturation, synaptic strengthening, and plasticity is PSD-95. Here we find that acute knockdown of PSD-95 in brain slice cultures by RNAi arrests the normal development of synaptic structure and function that is driven by spontaneous activity. Surprisingly, PSD-95 is not necessary for the induction and early expression of long-term potentiation (LTP). However, knockdown of PSD-95 leads to smaller increases in spine size after chemically induced LTP. Furthermore, although at this age spine turnover is normally low and LTP produces a transient increase, in cells with reduced PSD-95 spine turnover is high and remains increased after LTP. Taken together, our data support a model in which appropriate levels of PSD-95 are required for activity-dependent synapse stabilization after initial phases of synaptic potentiation.