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A link between obesity and cerebral health is receiving growing recognition. Here, we investigate in the frontal cortex and hippocampus the potential involvement of cholinergic markers in brain alterations previously reported in rats with obesity induced by diet (DIO) after long-term exposure (17 weeks) to a high-fat diet (HFD) in comparison with animals fed with a standard diet (CHOW). The obesity developed after 5 weeks of HFD. Bodyweight, systolic blood pressure, glycemia, and insulin levels were increased in DIO rats compared to the CHOW group. Measurements of malondialdehyde (MDA) provided lipid peroxidation in HFD-fed rats. Western blot and immunohistochemical techniques were performed. Our results showed a higher expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in obese rats but not the VAChT expression in the frontal cortex after 17 weeks of HFD. Furthermore, the acetylcholinesterase (AChE) enzyme was downregulated in HFD both in the frontal cortex and hippocampus. In the brain regions analyzed, it was reported a modulation of certain cholinergic receptors expressed pre- and post-synaptically (alpha7 nicotinic receptor and muscarinic receptor subtype 1). Collectively, these findings point out precise changes of cholinergic markers that can be targeted to prevent cerebral injuries related to obesity.