Despite significant advancements to understand of the neural circuitry involved in anxiety, the neurobiology of trait anxiety remains unclear. The rostral anterior cingulate cortex (rACC) and various pathways have been implicated in its regulation, making it a key to trait anxiety. The present study aimed to investigate the role of these neurotransmitter systems in the rACC in trait anxiety. Since trait anxiety is known to modulate state anxiety, we further investigated this relationship. Specifically, in Experiment I, we used animals with high trait anxiety; in Experiment II, we used animals with low trait anxiety; and in Experiment III, we used animals with medium trait anxiety. Before each behavioral assessment, drugs that either increased or decreased serotonergic (Fluoxetine or WAY-100635), GABAergic (Muscimol or Bicuculline), and glutamatergic (NMDA or Ketamine) neurotransmission in the rACC were administered, along with their respective controls. Additionally, in Experiment IV, all animals from the previous experiments were subjected to the Elevated Plus Maze (EPM) and Hole board (HB) test and evaluated without taking into account their trait anxiety levels. The results of the present study showed that, in Exp I, the modulation of the serotonergic, GABAergic and glutamatergic systems in the rACC decreased trait anxiety in highly anxious rats, while by submitting the animals to HB, the administration of fluoxetine increased state anxiety. In Exp II, the modulation of all systems increased trait anxiety in rats with low trait anxiety, whereas, in HB, state anxiety levels were increased with the administration of NMDA. In Exp III, only the modulation of the glutamatergic system, with NMDA, increased both trait and state anxiety levels. However, none of the evaluated neurotransmitter systems altered the state anxiety modeled in the EPM. Overall, the results of the present study provide new insights into the role of the neurotransmitter systems in the rACC in the regulation of trait anxiety and state anxiety.