The blood-brain barrier restricts the administration of drugs for neurological diseases. K16ApoE is an effective drug delivery carrier to deliver drugs across the blood-brain barrier, but it contains acute and high toxicity. The toxicity mechanism of K16ApoE must be revealed for clinical uses. Previous studies hypothesized that the toxicity mechanism was acetylcholinesterase inhibition in the brain. However, these studies used improper buffers in the AChE assay, further leading to anomalous results. Meanwhile, previous studies have not investigated the effects of K16ApoE on all the AChE-containing tissues and organs throughout the body. The previous dose design was also too narrow. Herein, we designed a more comprehensive and rational dose interval for K16ApoE, observed and recorded the mouse responses after receiving K16ApoE, and collected the brain, diaphragm, and serum to investigate the systemic K16ApoE effects on AChE. We also incubated purified AChE with K16ApoE in vitro. It could reveal the direct effect of K16ApoE on AChE without the influence of absorption and metabolism in vivo. The in vitro results demonstrated that K16ApoE inhibits rhAChE activity as the dose increases. However, the in vivo results demonstrated that K16ApoE does not affect tissue AChE activity in female mice. Therefore, we confirmed the AChE inhibitory effect of K16ApoE, but contrary to our hypothesis, AChE inhibition is not the toxicological mechanism of K16ApoE. We also recorded toxicological responses after the mice received K16ApoE, which would promote further toxicological investigation on K16ApoE.