Nature and wildlife observation is a popular activity that attracts many people. From the viewpoint of conservation ecology, it is important to know the effect of such human activity on the wildlife concerned. The giant flying squirrel (Petaurista leucogenys) is a recent, popular subject of observation in Japan. Yakuo-In Temple in Takao, Tokyo, is a renowned location for the observation of giant flying squirrel. However, the observation of this species at the temple only became popular in the mid1990s. Observation records made by a high school club revealed that giant flying squirrel in Yakuo-In departed their nests 30 min after sunset in 1987-1998; here, we report nest departure data for 2003-2004. Giant flying squirrel that lived in a highly visited nature-observation area departed their nests 60 min after sunset on average, whereas those that lived in a more secluded area departed their nests 30 min after sunset. Giant flying squirrel also departed their nests later when there were >20 observers near the nest. Thus, wildlife observation activity is responsible for the delay in the time of departure from the nest in giant flying squirrel.