Anxiety-related defensive behavior is controlled by a distributed network of brain regions and interconnected neural circuits. The dorsal raphe nucleus (DR), which contains the majority of forebrain-projecting serotonergic neurons, is a key brain region involved in fear states and anxiety-related behavior via modulation of this broad neural network. Evidence suggests that relaxin-3 neurons in the nucleus incertus (NI) may also interact with this network, however, the potential role of the NI in the control of anxiety-related defensive behavior requires further investigation. In this study, we examined the response of an anxiety-related neuronal network, including serotonergic neurons in the DR and relaxin-3-containing neurons in the NI, to administration of an anxiogenic drug and exposure to an aversive environment. We administered an anxiogenic dose of the adenosine receptor antagonist, caffeine (50 mg/kg, i.p.), or vehicle, to adult male Wistar rats and 30 min later exposed them to either an elevated plus-maze (EPM) or a home cage environment. Administration of caffeine and exposure to the EPM activated a broad network of brain regions involved in control of anxiety-like behaviors, including serotonergic neurons in the DR, as measured using c-Fos immunohistochemistry. However, only exposure to the EPM activated relaxin-3-containing neurons in the NI, and activation of these neurons was not correlated with changes in anxiety-like behavior. These data suggest activation of the NI relaxin-3 system is associated with expression of behavior in tests of anxiety, but may not be directly involved in the approach-avoidance conflict inherent in anxiety-related defensive behavior in rodents.