The rise of antimicrobial resistance has made necessary the increase of the antibacterial arsenal against multidrug-resistant bacteria. In this context, colistin has re-emerged as a first-line antibiotic in critical situations despite its nephro- and neuro- toxicity at peripheral level. However, the mechanism underlying its toxicity remains unknown, particularly in relation to the central nervous system (CNS). Therefore, this study aimed to characterize the molecular mechanisms underlying colistin-induced neurotoxicity in the CNS through a combination of in vitro and in vivo molecular studies along with several in vivo behavioral tests. Following colistin treatment, mice exhibited a significant reduction in body weight together with renal impairment, and locomotor dysfunction. Moreover, our results demonstrated that colistin disrupted the blood-brain barrier, inducing astrogliosis, and triggering apoptosis-related processes probably through the accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction. Further analysis on mice and primary neuronal cultures revealed that colistin administration altered neuronal plasticity by reducing the number of immature neurons in adult neurogenesis and altering the synaptic function through a reduction of the post-synaptic protein PSD95. All these alterations together finally lead to cognitive impairment and depression-like symptoms in mice. These findings provide novel insights into the mechanisms of colistin-induced neurotoxicity in the CNS, highlighting the need for careful monitoring of cognitive function in patients undergoing colistin treatment.