Animals alter their behaviour during habituation to novel environments. Echolocating bats exhibit remarkable flexibility in their acoustic signals to sense diverse microhabitats. Previous studies have described intra-individual variation in echolocation calls of bats in different environments, but few studies have systematically quantified these changes in detail. We investigated variation in echolocation call structure of the great leaf-nosed bat, Hipposideros armiger during habituation to a novel, captive environment. Echolocation calls of free-ranging bats were recorded in the natural habitat and in captivity over a three-week period. We found that bats exhibited significant changes in some call parameters following introduction to the novel captive environment, and some parameters changed continuously over time. We observed plasticity in peak frequency, pulse duration and pulse rate during the captive period. This suggests that variation in echolocation calls of bats in response to a novel captive environment is a progressive process, during which bats adjust echolocation call structure to habituate gradually to their surroundings.