Habitat fragmentation affects population density, resulting in a higher probability of extinction and lower rates of recolonization of habitat patches in some holarctic tree squirrels. Several local populations of the Japanese squirrel, Sciurus lis, seem to have decreased during the 1990s. We tested the hypothesis that the contracting distribution of Japanese squirrels is connected with habitat fragmentation via forest destruction for human land use. We investigated whether woodlot characteristics and degrees of woodlot isolation affected the presence of squirrels in suburban fragmented woodlots in western Tokyo. Japanese squirrels were present in 12 of 76 woodlots. Squirrel presence was not detected in all 23 woodlots east of a single north-south trending road within the study area. This result suggested that the road prevented squirrels from dispersing from the mountainous range west of the study area into eastern woodlots. In 53 woodlots west of the road, woodlot size and preferred habitat had positive effects on the probability of squirrel presence, whereas isolation variables did not affect its presence. We suspected that the habitat fragmentation via forest destruction for human land use might be one of the causes leading to the recent diminishing in local populations of Japanese squirrels.