Exposure to a stressful environment early in life can cause psychiatric disorders by disrupting circuit formation. Actin plays central roles in regulating neuronal structure and protein trafficking. Here, we found that neonatal chronic isolation inactivated ADF/cofilin, the actin depolymerizing factor, through stress-induced glucocorticoid hormone signaling. Inactivation of ADF/cofilin by neonatal isolation resulted in the increase of a stable actin fraction at spines in the developing rat barrel cortex and the juvenile medial prefrontal cortex. Furthermore, the reduced ADF/cofilin activity mediated the isolation-induced prevention of experience-driven synaptic delivery of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor. We identified a negative correlation between the amount of stable actin and the LTP-induced increase in surface AMPA receptors at individual cortical spines. Thus, the perturbation of ADF/cofilin function by neonatal isolation interferes with the experience-driven synaptic delivery of AMPA receptors by increasing stable actin at spines in multiple areas of the neocortex, potentially causing cortical dysfunction.