Abstract Macrophages can either promote or resolve inflammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine 5-HT). In fact, pro- and anti-inflammatory macrophages differ in the expression of serotonin receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF–primed monocyte-derived macrophages (M-MØ). 5-HT7 drives the acquisition of profibrotic and anti-inflammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT–related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antiparkinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT–mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B–specific antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics.