Soil microbial community dynamics during the spring freezing-thawing period could affect carbon and nutrient cycling in the subsequent growing season. During spring soil freezing-thawing period, we monitored temporal dynamics of soil microbial community in different soil substrates for Larix gmelinii forest using phospholipid fatty acids (PLFAs) as biomarkers every 3-7 days. The results were as followed: 1) the total PLFAs content, the PLFAs content and relative abundance of each soil microbial group, the ratio of Gram-positive bacteria to Gram-negative bacteria (G/G), the ratio of saturated PLFAs to unsaturated PLFAs (S/NS) and the ratio of bacteria to total fungi (fungi + arbuscular mycorrhizal fungi) (B/F) all varied among sampling times; 2) soil total organic carbon (TOC) and nitrogen (TN) were the main factors affecting soil microbial community in the early stage of soil freezing-thawing period; soil moisture, TOC and TN were the main driving factors in the middle stage of soil freezing-thawing period; soil microbes were affected by soil tempera-ture, moisture, TOC, TN and C/N in the late stage of soil freezing-thawing period; 3) the total PLFAs content, the PLFAs content and relative abundance of each soil microbial group (except the relative abundance of bacteria), B/F, G/G and S/NS all showed significant difference between soil substrates, and soil TOC, TN and C/N were the key determination factors. Soil temperature, moisture, and nutrient availability were the main factors affecting soil microbial community during the spring soil freezing-thawing period, but the degree of influence varied with the freezing-thawing stages and microbial groups.