Neuroanatomical findings revealed that CB1 cannabinoid and 5-HT3 receptors are coexpressed by a subtype of gamma-aminobutyric acid (GABA)ergic interneurons in the prefrontal cortex, hippocampus, and basolateral amygdala, three brain regions that are crucial for the control of anxiety. In these regions, serotonergic inputs increase GABA release through 5-HT3 receptors, the phenomenon being retrogradely controlled by cannabinoid neurotransmission. This suggests a functional interaction between 5-HT3 neurotransmission and CB1 signaling. In a first attempt to investigate the behavioral relevance of these interactions, we studied the effects of the selective 5-HT3 agonist 1-(m-chlorophenyl)-biguanide (mCPBG), on plus-maze behavior in NMRI, CD1 wild type, and CB1-KO mice. The genetic disruption of CB1 receptors consistently increased anxiety. This effect was significantly decreased by the 5-HT3 agonist, mCPBG. The dose-response curve was bell-shaped. Surprisingly, mCPBG did not affect the behavior of CD1 wild type and NMRI mice. We hypothesize that in the aforementioned regions, 5-HT3 activation decreases anxiety by promoting GABA release, but this effect is dampened by CB1 signaling. The disruption of CB1 receptors in CB1-KOs released GABA neurons from retrograde inhibition and made the effects of 5-HT3 stimulation conspicuous. Altogether, our findings reveal a functional interaction between 5-HT3 neurotransmission and CB1 signaling. Besides this interaction being an interesting aspect of anxiety control, it may also explain the notoriously inconsistent effects of 5-HT3 ligands on anxiety. If 5-HT3 neurotransmission and CB1 signaling interact, the anxiety-related effects of 5-HT3 ligands may depend on species, strain, and situation-related differences in both 5-HT3 and CB1 receptor expression and function.