Dendritic spines are major sites of excitatory synapses in the brain and display rapid motility, which is believed to be important for synapse formation and plasticity. Spine morphology was previously shown to be regulated by the Rho GTPases Rac1 and RhoA. Here, we analyzed the roles of Rac1 and a downstream effector of RhoA, Rho kinase, in controlling spine morphogenesis and their effects on spine motility and stability. Blockade of Rac1 induced long, thin spines and inhibited spine head growth, morphing, and stability. Spine head growth was more severely affected in mature spines. On the other hand, inhibition of Rho kinase induced new, long spines and protrusive motility. These data demonstrate that Rac1 and RhoA/Rho kinase pathways regulate different aspects of spine morphology, motility, and stability and presumably also different aspects of synaptic functions. Moreover, our data show that there are two different types of spine motility: protrusive motility and head morphing, which are differentially regulated by Rac1 and Rho kinase. We propose that these two different types of spine motility serve different functions in synaptogenesis and synapse maturation.