Birds have the ability to assess the risk of predation in their environment and adjust their antipredation strategies based on this risk information. However, whether nest site selection has effect on subsequent nest defence behaviour has not been studied. In this study, we aimed to determine whether the Japanese tit (Parus minor) exhibits a nest-box hole size preference and whether the entrance hole sizes of nest boxes influence the nest defence behaviour of tits. We hung nest boxes with three different entrance hole sizes (diameters: 6.5cm, 4.5cm and 2.8cm) in our study sites and investigated which nest boxes were occupied by tits. In addition, by using dummy-presentation experiments, we observed the nest defence behaviours of tits that nested in boxes with 2.8cm and 4.5cm entrance holes towards common chipmunks (Tamias sibiricus, a small nest predator able to enter these holes) and Eurasian red squirrels (Sciurus vulgaris, a large nest predator unable to enter the 2.8cm entrance hole). The tits that bred in nest boxes with 2.8cm entrance holes exhibited more intense nest defence responses to chipmunks than to squirrels. In contrast, the tits that bred in nest boxes with 4.5cm entrance holes exhibited similar nest defence responses to chipmunks and squirrels. Additionally, Japanese tits that bred in nest boxes with 2.8cm entrance holes exhibited more intense behavioural responses to chipmunks than those that bred in nest boxes with 4.5cm entrance holes. Our results suggested that Japanese tits prefer to occupy nest boxes with small holes for breeding and that nest-box characteristics influenced their nest defence behaviour.