An extract from aerial parts of Ajuga nipponensis Makino was examined by high performance liquid chromatography for minor ecdysteroids. Along with the compounds already reported, namely cyasterone, ajugasterone C, cyasterone 22-acetate and 22-dehydrocyasterone, the presence of three additional bands with the expected ecdysteroid-like UV absorption was observed. The structures of the isolates were unambiguously elucidated based on extensive NMR spectral studies (one and two-dimensional experiments) and pointed out three new phytoecdysteroids. One of the new compounds, 22-dehydrocyasterone 2-glucoside is just the second example of a C-2 glucosyl derivative. The other two compounds displayed hemiacetal functions in the side chain, one unprecedented, and were named ajugacetalsterone A and B.