No studies have examined the effect of experimental warming on the microbial biomass and community composition of soil in agricultural ecosystem on the Qinghai-Tibet Plateau. Thus it is unclear whether the influences of experimental warming on microbial communities in soil are related to warming magnitude in croplands on this Plateau. This study performed warming experiment (control, low- and high-level) in a highland barley system of the Lhasa River in May 2014 to examine the correlation between the response of microbial communities in soil to warming and warming magnitude. Topsoil samples (0–10 and 10–20 cm) were collected on September 14, 2014. Experimental warming at both low and high levels significantly increased soil temperature by 1.02 °C and 1.59 °C, respectively at the depth of 15 cm. Phospho lipid fatty acid (PLFA) method was used to determine the microbial community in soil. The low-level experimental warming did not significantly affect the soil's total PLFA, fungi, bacteria, arbuscular mycorrhizal fungi (AMF), actinomycetes, gram-positive bacteria (G⁺), gram-negative bacteria (G^–), protozoa, the ratio of fungi to bacteria (F/B ratio), and ratio of G⁺ to G^– (G⁺/G^– ratio) at the 0–10 and 10–20 cm depth. The low-level experimental warming also did not significantly alter the composition of microbial community in soil at the 0–10 and 10–20 cm depth. The high-level experimental warming significantly increased total PLFA by 74.4%, fungi by 78.0%, bacteria by 74.0%, AMF by 66.9%, actinomycetes by 81.4%, G⁺ by 67.0% and G^– by 74.4% at the 0–10 cm depth rather than at 10–20 cm depth. The high-level experimental warming significantly altered microbial community composition in soil at the 0–10 cm depth rather than at 10-20 cm depth. Our findings suggest that the response of microbial communities in soil to warming varied with warming magnitudes in the highland barley system of the Lhasa River.