The K16ApoE peptide has been demonstrated to deliver a supraphysiological level of protein therapeutics to the brain and further increase the life-span of mice with a lysosomal storage disorder (LSD). If successfully developed, K16ApoE would provide new treatments for LSD and many other neurological diseases. However, while the K16ApoE can cross the blood-brain barrier, data indicates a toxic response associated with it. The mechanism of toxicity must be resolved for further clinical translation. The toxic response towards the peptide was hypothesized to be induced by inhibition of acetylcholinesterase (AChE) activity at neuro-muscular junction. Here, the dose-response analysis between AChE and K16ApoE was conducted in both female and male mice. Results demonstrated that AChE activity was significantly reduced with increasing dose of K16ApoE except for the mid-dose where a dramatic increase in AChE activity was observed. Also, obvious difference in response to K16ApoE was shown when considering the influence from sex and body weight. Though the statistical analysis of the dose response and survival ratio suggested that AChE is not the primary mechanism of action for the acute toxicity of K16ApoE, the complex inhibition/stimulation response of AChE indicated that this enzyme must play a role in the toxicity of the peptide.