Soil macrofauna under microhabitat (soil surface, stone pit, stone gully) in karst region of Huajiang river valley is studied, and community structure of soil animal, spatial distribution and biomass are used in order to prove the microhabitat impact to soil animal in the paper. The results show that there are 276.7 soil macrofauna that belong to 3 phylums, 7 classes, 24 species under microhabitat in limestone in southwest Guizhou being discovered. The predominant group of ants accounts for the total of 56.75%. Diversity of soil surface, stone gully and stone pit is 1.199, 1.592 and 1.962; evenness index of soil surface, stone gully and stone pit is 0.616, 0.989 and 1.095; dominance of soil surface, stone gully and stone pit is 0.545, 0.353 and 0.245 in soil macrofauna community structure. Correlation analysis with spss13.0 shows that there is no remarkable influence to individual number and group number by different microhabitat and soil layer with F-value being 0.834 and 1.226 respectively, but remarkable influence to the soil animal biomass by microhabitat with F-value being 3.844. In three kinds of microhabitats, stone gully is advantageous to soil animal survival. The harsh microhabitat and sole species lead to the weaken of soil animal biomass and community.