Males of the Strawberry Poison Frog (Oophaga pumilio) use acoustic signals during courtship and territorial interactions. In these contexts, spectro-temporal characteristics of the calls provide information on body size, territory quality, and dominance to conspecifics. Previous research on this species has associated low-rate territorial calls produced by resident males with greater aggressiveness. However, how variation in call rate of intruding males affects the response of the territorial males has not been tested experimentally. We evaluated the effect of different call-rate stimuli (low, average, and high call rate) on the acoustic and behavioral responses of territorial males of the Strawberry Poison Frog using playback experiments to simulate territorial male intrusions. We found that territorial males lowered their call rate in response to playbacks with high and average call rate. In addition, territorial males approached three times more, closer, and faster to the average and low call-rate playback stimuli than to high call-rate stimulus. Males also responded, vocalizing faster to the average and low call-rate playback stimuli than to high call-rate stimulus. Our results suggest that different call rates correlate with different levels of aggressiveness in males of the Strawberry Poison Frog. Therefore, the observed behavior supports the hypothesis that a low call rate indicates higher aggressiveness in male–male interactions.