The suitability of a soil proteome analysis based on previous cell extraction by gradient centrifugation was tested in semiarid soils with distinct edaphic properties and microbial biomass after enrichment with carbon and nitrogen. A sandy loam soil with low organic carbon content reached higher microbial biomass (estimated by PLFAs) after stimulation with nutrient sources (glucose and proline) than a naturally rich soil. However, the extractability of soil microbial cells was higher in a poor soil with high electrical conductivity probably due to the high saline content. The number of identified proteins in the poor soils reached 71 with proteins related to energy processes, transport and nucleic acid metabolism representing the highest percentage. High organic carbon content negatively influences cell extraction and protein separation and analysis. Soil texture and/or salinity might be related to the expression of proteins involved in the removal of reactive oxygen species (ROS) such catalase and superoxide dismutase (SOD) under active metabolism and microbial biomass development