A single exposure to stress can induce functional changes in neurons, potentially leading to acute stress disorder or post-traumatic stress disorder.In this study, we used in vivo wide-field optical mapping to simultaneously measure neural calcium signals and hemodynamic responses over the whole cortical area. We found that cortical mapping to whisker stimuli was altered under acute stress conditions. In particular, callosal projections in the anterior cortex (primary/secondary motor, somatosensory forelimb cortex) relative to barrel field (S1BF) of somatosensory cortex were weakened. On the contrary, the projections in posterior cortex relative to S1BF were mostly unchanged or were only occasionally strengthened. In addition, changes in intra-cortical connection were opposite to those in inter-cortical connection. Thus, the S1BF connections to the anterior cortex were strengthened while those to the posterior cortex were weakened. This suggests that the well-known barrel cortex projection route was enhanced. In summary, our in vivo wide-field optical mapping study indicates that a single acute stress can impact whole-brain networks, affecting both neural and hemodynamic responses.