Health sciences have recently discovered the medical uses of nandrolone decanoate (ND), an androgenic anabolic steroid (AAS), and reported its use in human and animal patients. Clinical evidences suggest that the AAS excess may affect the cholinergic system, which is responsible for several vital functions like learning, memory, and the organization of the movements. Thus, our aim is to research the subchronic effect of ND when administered in varying doses on the acetylcholinesterase (AChE) activity in these brain structures: cerebellum (CE), hippocampus, striatum (ST), and cortex of adult rats. We used 36 male Wistar rats, which were divided into six groups (n=6). The groups were divided into: G1-control (physiologic solution), G2-diluents control (only an oleaginous vehicle of vegetal origin-olive oil), G3-0.42 mgkg −1 of ND, G4-1.8 mg kg −1 of ND, G5-4.6 mgkg −1 of ND, and G6-10.0 mg kg −1 of ND. We applied the doses once every week during a 3-week period. The values obtained demonstrated a significant increase in the AChE activity (referring to ST and CE for the 4.6 and 10.0 mgkg −1 doses of ND). The ND causes increase in AChE activity, which could impair neurotransmission and cholinergic modulation.