Acetylcholine (ACh), a quaternary ammonium cation, is known as one of the itch inducer in atopic dermatitis (AD), an inflammatory skin disease with intense itching. Previous research has reported accumulation of ACh in lesional site of AD patients. Generally, ACh is metabolized by cholinesterase (ChE). Therefore, one of the causes of ACh accumulation may be the suppression of ChE activity. Increased levels of the multifunctional bioactive sphingolipid sphingosylphosphorylcholine (SPC) have also been detected in AD. Since SPC possesses a quaternary ammonium cation, like ACh, it is possible that SPC affects the activity of ChE catalyzing ACh metabolization. We investigated whether SPC influences the activity of ChE by performing enzymatic analysis of ChE in the presence of SPC. We found that SPC strongly suppressed acetylcholinesterase (AChE) activity, but the suppression of butyrylcholinesterase by SPC was quite low. The Michaelis constant (K_m) of AChE in the presence of SPC increased, and the maximum velocity (V_max) decreased, indicating that SPC acts as mixed-type inhibitor for AChE. The analysis of SPC analogs clarified the importance of both the quaternary ammonium cation and the carbon chain length of SPC for the AChE inhibitory effect and showed that SPC was unique in AChE inhibition among the sphingolipids in this study. These findings indicate a novel function of SPC on AChE inhibition. Thus, the inhibition activity of SPC may be a factor in the increase of ACh in AD.