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Human red blood cell acetylcholinesterase (RBC-AChE) activity is valuable for detecting potential exposure to cholinesterase inhibiting substances (CIS). A reliable population-based RBC-AChE activity reference range is critical for early and massive clinical and occupational toxicology screening. Previous published studies were often limited to small numbers of subjects, various testing methods, and crude statistical data analyses. We tested 4818 adult subjects with a well-established 17-minute modified Michel method over a 2-year period. We conducted a retrospective data analysis and systematically investigated on the influences to testing values from gender, age, age group, and their combinations and interactions. No significant difference was observed in the testing values between males (mean, medium, interquartile range = 0.76, 0.76, 0.71–0.80 ΔpH/h, respectively) and females (mean, medium, interquartile range = 0.76, 0.76, 0.71–0.81 ΔpH/hour, respectively), when gender was the only factor considered (p = 0.7238). However, with age progression, male testing values exhibited a consistent upward trend, while females did not show any clear patterns. Linear regression analysis of the data revealed that gender, age, and age group more or less affected testing values either as independent variables or with their combinations and interactions. However, more potential factors need to be included to achieve better testing value predictions. We recommend the toxicological testing community to adopt a new set of age group specific RBC-AChE activity reference ranges for males (0.68–0.80, 0.69–0.81, 0.70–0.83, 0.71–0.84, and 0.73–0.87 ΔpH/h for 18–29, 30–39, 40–49, 50–59, and ≥60 years old, respectively) while keeping the current reference range (0.63–0.89 ΔpH/hour) for females. [ABSTRACT FROM AUTHOR]