Since structural changes in dendritic spines are tightly coupled to alterations in synaptic transmission, modifications in the spine morphology are considered an important component of the molecular basis of learning and memory. The morphological changes in spines are controlled by rearrangement of the actin cytoskeleton and occur differently in various types of neurons. However, how the actin dynamics are regulated in a neuronal cell-type specific manner remains largely unknown. Here, we demonstrate that Fhod3, a member of the formin family of F-actin organizing factors, regulates dendritic spine morphogenesis in subpopulations of pyramidal neurons in the mouse cerebral cortex. We show that Fhod3 is expressed only in layer II/III and V of cerebral cortex in specific area. Synaptosomal fractions and immunofluorescence analyses exhibit Fhod3 accumulation in postsynaptic spines. Even though Nestin-Cre-mediated Fhod3 knockout mice show no gross phenotype, we find atypical dendritic spines morphology of pyramidal neurons in presumptive Fhod3-positive areas. In primary cultures prepared from the Fhod3-null cortex, atypical dendritic spines morphology are found in Fhod3-promoter-active neurons, a particular subpopulation of pyramidal neurons, and not in Fhod3-promoter-negative pyramidal neurons. We conclude that Fhod3 contributes to dendritic spine morphogenesis in particular subpopulations of pyramidal neurons.