The rodent somatosensory barrel cortex is an ideal model for studying the impact of sensory experience on developing brain circuitry. To examine whether and how interference with sensory perception in the early postnatal period can affect the development of synaptic networks in this system, we took advantage of a transgenic mouse strain expressing the yellow fluorescent protein in layer 5B pyramidal neurons of the somatosensory cortex. By using ex vivo confocal imaging, we first demonstrate a cortical‐layer‐specific increase in the number of dendritic spines during postnatal development on apical dendritic shafts of these cells extending up to cortical layer 1. Next, by performing bilateral whisker trimming at distinct developmental stages, we show that disruption of sensory perception before postnatal day 20 impairs dendritic spine development in apical dendritic segments within layers 1 and 2/3 but not in layer 4. The whisker trimming‐induced decrease in dendritic spine density during this period is accompanied by a highly significant decrease in dendritic spine head diameter. Finally, we also show that these whisker trimming‐induced morphological alterations of dendritic spines during the early postnatal period are no longer detectable in adult animals. Altogether, these findings further emphasize the important role of sensory activity in synaptic network assembly in the developing barrel cortex. They also support an as yet unidentified structural mechanism that might contribute to the layer‐ and cell‐type‐specific physiological effects of whisker trimming during the early postnatal period. J. Comp. Neurol. 518:1711–1723, 2010. © 2009 Wiley‐Liss, Inc.