<p>The CXCL12/CXCR4/ACKR3 axis is essential for brain development and homeostasis. Dysregulation of CXCL12 or its receptors has been associated with various CNS disorders related to neurodevelopmental defects, neuroinflammation, neurodegeneration and brain tumors. This signaling axis represents an attractive therapeutic target, and large efforts have led to the development and use of specific pharmacological modulators for brain diseases. While pharmacological modulation of CXCR4 has extensively been studied in the last decades, targeting ACKR3 has only recently emerged as a critical component of the CXCL12 signaling network, driving growing interest in the development of ACKR3-specific modulators.</p><p>This review synthesizes CXCL12-induced distinct signaling mechanisms downstream of the receptors: G protein-dependent for CXCR4, and β-arrestin-biased for ACKR3, which acts as a scavenger. We also describe the expression and function of CXCL12, CXCR4 and ACKR3 under physiopathological conditions in the CNS. We exhaustively depict the current status of specific modulators of CXCL12 and its receptors, from in vitro testing to preclinical studies and clinical trials, through exploring various neuropathological contexts, e.g. multiple sclerosis, Alzheimer’s and Parkinson’s diseases, amyotrophic lateral sclerosis or cancer. We highlight how pharmacological modulation of one or another triad component drives context-dependent outcomes, and we point out the limitations that should carefully be addressed in the future, to advance CXCL12/CXCR4/ACKR3 targeting approaches for CNS pathologies.</p>