Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disorder, characterized by β-amyloid plaques, neurofibrillary tangles, and loss of cholinergic neurons. Butyrylcholinesterase (BChE) inhibition is a critical strategy for the treatment of AD since BChE causes inactivation of neurotransmitter acetylcholine and has positive effects on promoting the formation of βamyloid fibrils. Our previous studies showed that various phenothiazine-derived compounds such as thionine and toluidine blue O (TBO) cause a potent inhibition of human cholinesterases. TBO was also found to affect amyloid precursor protein processing in-vitro and in-vivo models of AD. In this study, it was aimed to determine the inhibitory effect of 1,9-dimethyl-methylene blue (DMMB), a phenothiazine-derived compound, on human plasma BChE and explore its inhibitory mechanism. The inhibition of human BChE was assessed by the colorimetric method of Ellman using butyrylthiocholine as substrate and 0-0.375 µM of DMMB. The kinetic findings showed that DMMB acts as a linear mixed-type inhibitor of human BChE with Ki value of 23 ± 0.004 nM and α= 3.6 ± 1.6. In conclusion, DMMB, which is a potent inhibitor effective at nM level, may be helpful in designing new cholinesterase inhibitors for the treatment of AD.