Organophosphate pesticides as diazinon disrupt the neuroimmune communication, affecting the innate and adaptive immune response of the exposed organisms. Since the target molecule of diazinon is typically the acetylcholinesterase enzyme (AChE), the existence of a non-neuronal cholinergic system in leukocytes makes them susceptible to alterations by diazinon. Therefore, the aim of this work was to evaluate the activity of AChE, acetylcholine (ACh) concentration, and the expression of nicotinic ACh receptors (nAChR) and muscarinic ACh receptors (mAChR) in spleen mononuclear cells (SMNC) of Nile tilapia (O. niloticus) exposed in vitro to diazoxon, a diazinon metabolite. SMNC were exposed in-vitro to 1 nM, 1 μM, and 10 μM diazoxon for 24 h. The enzyme activity of AChE was then evaluated by spectrophotometry, followed by ACh quantification by ultra-performance liquid chromatography. Finally, mAChR and nAChR expression was evaluated by RT-qPCR. The results indicate that AChE levels are significantly inhibited at 1 and 10 μM diazoxon, while the relative expression of (M3, M4, and M5) mAChR and (β2) nAChR is reduced significantly as compared against SMNC not exposed to diazoxon. However, ACh levels show no significant difference with respect to the control group. The data indicate that diazoxon directly alters elements in the cholinergic system of SMNC by AChE inhibition or indirectly through the interaction with AChR, which is likely related to the immunotoxic properties of diazinon and its metabolites. Display omitted •OPs are capable of disrupting the cholinergic system of SMNC in Nile tilapia.•AChE activity is inhibited in SMNC exposed in-vitro to 1 and 10 μM of DXN.•DXN alters the expression of mAChR and nAChR in SMNC of Nile tilapia.•DXN inhibits AChE but also alters " non-conventional " targets such as AChR.