Regulation of AMPA receptor (AMPAR) membrane trafficking is critical for synaptic plasticity, as well as for learning and memory. However, the mechanisms of AMPAR trafficking in vivo remain elusive. Using in vivo two-photon microscopy in the mouse somatosensory barrel cortex, we found that acute whisker stimulation led to a significant increase in the intensity of surface AMPAR GluA1 subunit (sGluA1) in both spines and dendritic shafts and a small increase in spine size relative to prestimulation values. Interestingly, the initial spine properties biased spine changes following whisker stimulation. Changes in spine sGluA1 intensity were positively correlated with changes in spine size and dendritic shaft sGluA1 intensity following whisker stimulation. The increase in spine sGluA1 intensity evoked by whisker stimulation was NMDA receptor dependent and long lasting, similar to major forms of synaptic plasticity in the brain. In this study we were able to observe experience-dependent AMPAR trafficking in real time and characterize, in vivo, a major form of synaptic plasticity in the brain.