Ca²⁺/Calmodulin-dependent kinase II (CaMKII) is one of the most important signaling molecules for long-term potentiation and associated spine enlargement underlying learning and memory. Here, to understand the function of CaMKII for synaptic plasticity, we developed genetically encoded light-inducible CaMKII inhibitor and photo-activatable CaMKII by using LOV2 derived from phototropin1. We applied these newly developed optogenetic tools for the study of structural plasticity of single dendritic spines by using 2-photon fluorescence microscope and glutamate uncaging, and found that 1) ~60 s of CaMKII activation is sufficient for inducing transient and sustained spine enlargement, 2) CaMKII activation alone is sufficient for triggering structural plasticity. In addition, using 2-photon fluorescence lifetime imaging microscopy, we visualized the activity of CaMKII downstream molecules, Cdc42 and RhoA which are the regulator of actin polymerization, to understand the spatiotemporal dynamics of these molecules during structural plasticity. And, we found that Cdc42 and RhoA were rapidly activated in the stimulated spine, and were then followed by a phase of persistent activation lasting more than 30 min. Furthermore, the inhibition of the RhoA pathway preferentially inhibited the initial spine growth, whereas the inhibition of the Cdc42 pathway blocked the maintenance of sustained structural plasticity. Thus, RhoA and Cdc42 relay transient CaMKII activation to synapse-specific, long-term signaling required for spine structural plasticity.