Although several studies have reported inter- and intra-individual variation in the echolocation pulses of big brown bats, relatively few have explored its function in the context of courtship and mating. In this study, we tested for sex and seasonal differences in the echolocation pulses of male and female big brown bats, and related these differences to mating activity. We recorded the echolocation pulses of adult female and male big brown bats in a controlled laboratory environment during the mating and three non-mating seasons. Factor analysis was used to reduce the number of call variables into two principle components (PC1 and PC2). Call components related to PC2 were significantly sexually dimorphic in the mating season. However, no call components were significantly sexually dimorphic in the non-mating season. In addition, we found a significant correlation between PC1 and the mating score of male bats. There was no significant correlation between principle components and the mating score of female bats. Our results provide additional support for the current literature that suggests a communicative function for bat echolocation pulses. Furthermore, it suggests that differences in the echolocation pulses of male and female bats may be important in the mating activity of this group.