Background: Impaired sympathetic/parasympathetic response, expressed by elevated Acetylcholinesterase (AChE) is associated with obesity, metabolic syndrome and inflammation. However, the association between morbid obesity and AChE and the changes in cholinergic tone following bariatric laparoscopic sleeve gastrectomy (LSG) surgery-induced weight reduction were never analyzed. Methods: Two studies are presented; the first (the “apparently healthy cohort”) was a cross-sectional study and the second (the “LSG cohort”) was a prospective-cohort study with 12 months of follow-up. The “apparently healthy cohort” included 1450 apparently healthy participants who volunteered to the Tel-Aviv Medical Center Inflammation Survey (TAMCIS) during a routine annual checkup visit. The “LSG cohort” included 77 morbid obese patients before and at 3, 6, and 12 months following LSG surgery. Main outcomes included anthropometric measurements, Hemoglobin A1c (HbA1C), serum AChE, insulin test and Homeostasis Model Assessment (HOMA). Results: Among the TAMCIS participants, serum AChE activity increased with BMI in a dose-dependent manner until it reached a peak level at BMI of 30–35 kg/m², followed by a plateau. Following LSG, a significant decrease in AChE activity between baseline and 12 months post-surgery was found for men, but not for women (−122.2 ± 135.3, P < 0.001 vs. −21.8 ± 120.5, P = 0.258 nmol substrate hydrolyzed/min per ml, respectively). The reduction in AChE activity was negatively correlated with %excess weight loss (EWL) and positively correlated with %body fat reduction at 12 months post-surgery among women (r = −0.329, P = 0.034 and r = 0.350, P = 0.023, respectively). In men, AChE activity reduction was positively correlated with the HOMA reduction (r = 0.358, P = 0.048). Conclusions: Obesity-related AChE resistance phenotype may be reversed following LSG and correlates with metabolic outcomes. Further long-term studies will be needed to validate and evaluate the beneficial effect of AChE reduction post bariatric surgery.