Chemical constituents in ethyl acetate and butanol fractions of ethanol extracts from Acorus tatarinowii were separated by column chromatography. Bufo skeletal muscle fatigue model was established to study the anti-fatigue activity of separated compounds. Five compounds were separated and identified by spectroscopic analysis as acoramone(1),cycloartenone(2),2,4,5-trimethoxyl-2'-butoxy-1,2-phenyl propandiol(3),5-hydroxymethyl furfural(4), and 5-butoxymethyl furfural(5). Compound 3 was a new compound, and compounds 2 and 5 were separated from this plant for the first time. Compound 4 exhibited a notable anti-fatigue activity.