Echolocation calls produced by bats contain information on the emitter that could be used in social interactions. Nevertheless, little is known about the whether such information actually is being used by bats. Here we tested the ability of two horseshoe bat species (Rhinolophus mehelyi and R. euryale; n = 20 each) to discriminate conspecific male from conspecific female echolocation calls and vice versa. Behavioural data from a habituation-discrimination paradigm revealed that both species were able to recognize the sex of conspecifics from their calls, providing evidence that cues on the sex of the emitter are encoded in echolocation signals. While our data support that the sex-specific acoustic cues are not (just) the horseshoe bats' constant-frequency call part, their nature remains enigmatic. This is the second study to show that bats can recognize the caller's gender from echolocation calls and the first to do so for ‘high-duty-cycle bats’; those that use calls with a prominent constant-frequency (CF) component.