<p>Chronic pain induces long-lasting changes in the anterior cingulate cortex (ACC) activity that contribute to the development of comorbid mood disorders. However, how such alterations propagate throughout the ACC connectome remains to be elucidated. Here, we aimed to study the role of the ACC neurons projecting to the lateral habenula (LHb) (ACC_→LHb) in chronic pain-induced depression (CPID). CPID was induced using sciatic nerve cuffing in male C57BL/6J mice, and anxiodepressive-like behaviors were evaluated using a battery of behavioral tests. Fiber photometry was used to study the Ca²⁺ dynamics in the ACC, LHb, and ACC_→LHb neurons_. We combined viral Translating Ribosome Affinity Purification (vTRAP) and RNA sequencing to study the molecular alterations in the ACC_→LHb neurons. Finally, we used an optogenetic approach to study the functional role of this pathway in CPID. Our results confirmed a functional connectivity between the ACC and LHb and demonstrated that this connection plays a critical role in emotional processing. Activation of ACC_→LHb neurons elicited Ca²⁺ responses in the LHb and induced anxiodepressive-like behaviors in naive mice. Cell-type specific transcriptomic analysis revealed that CPID altered the expression of genes involved in neuronal excitability, such as genes related to sphingolipid metabolism, glycophospholipid,s and Ca²⁺ channels in ACC_→LHb neurons. Interestingly, inhibition of this hyperactivity alleviated chronic pain- but not stress-induced anxiodepressive-like behaviors, demonstrating that the ACC_→LHb pathway selectively contributed to nerve-injury induced emotional dysregulation. These results reveal that hyperactivity of the neuronal pathway linking the ACC to the LHb is essential for CPID in male mice.</p>