Alzheimer's Disease (AD) is a neurodegenerative disorder that is associated with acetylcholine deficiency in the brain, oxidative stress and inflammatory processes. One of the treatments of AD consists in the inhibition of acetylconesterase. The flavanoid vitexin (8-C-β-D-glycosylapigenin), present on the leaves of Jatropha mutabilis (Euphorbiaceae). Thus, the present study evaluates the inhibitory activity of vitexin isolated from Jatropha mutabilis, against the enzyme acetylcholinesterase (EACh). In the inhibition test of EACh, the Ellman methodology was performed. In silica gel chromateplates, caffeine (as positive pattern) and vitexin (isolated in 5 concentrations) were applied. Then, Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid)/ATCI - acetylthiocholine iodide) was sprayed onto the plate, and after drying, the enzyme for revelation. For the quantitative test, 100 μL of samples (1,0 - 0,0625 mg mL-1) in buffer were mixed with 100 μL of EACh 0,22 U mL-1 and 200 μL of buffer (50 mM Tris-HCl, pH 8, 0,1% bovine serum albumin and 500 μL DTNB. The white reaction was prepared by substitution of EACh. The reaction was monitored by 5 min at 412 nm. The mean inhibitory concentration values (CI50) were obtained by plotting Log-Probit. On the CCD plate, white spots were observed on the yellow chromate plate, indicative of inhibition of the EACh by caffeine and vitexin at all concentrations. In the quantitative test there was a concentration-dependent inhibition with a percentage of 74,42; 60,11; 59,45; 45,36; 41,43% at concentrations of 1,0; 0,5; 0,25; 0,125 and 0,00625 mg/mL, respectively. The IC50 found was 88,38 μg/mL. Vitexin showed strong inhibition of the enzyme acetylcholinesterase in in vitro tests, when compared to other flavonoids obtained from plants with reported neuroprotective activity.