Actin cytoskeleton is composed of functionally distinct pools of filamentous (F)-actin defined by their regulatory machinery and dynamics. Although these networks may compete for actin monomers and regulatory factors1-4, the interaction between them remains poorly understood. Here, we show that disruption of the labile F-actin pool in neurons by limited actin depolymerization5,6 unexpectedly triggers rapid enhancement of the F-actin content at the dendritic spine. Long-term blockade of NMDA-type receptors decreases spine actin polymerization, which is specifically restored by the labile pool ablation. Increase in the spine actin is triggered by blockade of formin-induced actin polymerization in a manner dependent on Arp2/3 complex activity. Finally, limited actin depolymerization increases F-actin levels in a cultured cell line, suggesting the generality of the two-tiered actin dynamics. Based on these findings, we propose a model whereby the labile pool of F-actin controlled by formin restricts the polymerization state of the Arp2/3-regulated stable spine actin, suggesting a feedback principle at the core of cytoskeletal organization in neurons.