Altered sensory input can change response properties in the visual cortex. How this is implemented at the level of individual synapses is not well understood. In this study we explore whether changes of neuronal response properties induced by monocular deprivation (MD) correlate with alterations of the number and size of dendritic spines on pyramidal neurons in the visual cortex of adult mice. To address this question, we combined intrinsic signal imaging and two-photon microscopy and anaesthetized mice implanted with cranial windows. This approach allowed us to monitor functional changes of eye-representation in the binocular part of the visual cortex, as well as to image repeatedly the apical dendrites of GFP-labelled neurons before, during and after MD. We found that a brief period of MD robustly altered the balance of dendritic spine turnover in layer 5 neurons in the binocular visual cortex of adult mice. More spines were added during MD than under baseline conditions. In contrast, the rate of spine elimination did not change during MD, leading to an increased spine density. This effect was cell type specific, as we did not observe similar changes in layer 2/3 neurons. The additional spines in the apical dendrites of layer 5 cells might form a structural correlate of the increase in non-deprived eye response strength, which is the main effect seen after MD in adult mice. Restoring binocular vision returned spine dynamics to baseline levels, but absolute spine density remained persistently elevated. Importantly, spine addition did not increase again when the same eye was closed for a second time. This absence of structural plasticity after a second MD episode contrasts with a robust and even faster change of eye-specific responses after repeated MD. Thus, dendritic spines added during the first monocular deprivation experience may provide a structural basis for subsequent functional shifts. It has been shown that spine size and synapse strength are correlated. Therefore, as a first step towards characterizing the functional significance of the newly formed spines, we followed the size of individual spines that appeared during MD. As a measure for spine size we used integrated spine brightness. New MD induced spines became larger during deprivation, and they were of similar size as newly formed spines in control animals. However, spines that appeared during MD shrank again after binocular vision had been restored and, importantly, their size increased again during the second MD. In contrast, persistent new spines in control animals grew initially and then stabilized, but on average did not shrink after their appearance. Thus, size changes of MD-induced spines correlate with the strengthening, weakening and re-strengthening of non-deprived eye responses during MD, recovery and second MD, respectively, thereby supporting the idea that these new spines are functionally important as they are might bear synapses whose strength is modulated by visual experience. Overall, these results provide a strong link between functional plasticity and specific synaptic rearrangements, revealing a mechanism of how prior experiences could be stored in cortical circuits.