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Glyphosate, a broad-spectrum herbicide used for the non-selective control of weeds, inhibits 5enolpyruvylshikimate-3-phosphate synthase, a key enzyme in the synthesis of aromatic amino acids in the shikimic acid pathway in plants, fungi and bacteria, thus impairing the synthesis of proteins required for various life processes. Soybean genetically engineered to be glyphosate resistant (GR or Roundup Ready, RR) represents the most cultivated transgenic crop globally, including Brazil. There are concerns about the effects of RR transgenic soybean and of glyphosate on soil microbial communities and their functioning. Our study was designed to detect changes in soil microbial biomass-carbon (MB-C) and-nitrogen (MB-N) and in enzyme activities [beta-glucosidase (GLU) and acid phosphatase (PHO)] in a large set of field trials performed at six sites in Brazil for two cropping seasons. We evaluated the effects of the RR transgene, glyphosate and weed management (RR soybean + glyphosate vs. conventional soybean + conventional herbicides), with three pairs of nearly isogenic soybean cultivars evaluated per site. Soils were sampled from the 0-10 cm layer, between cropped lines, during the cropping seasons 2004/2005 and 2005/2006, at the R2 stage of soybean growth. Univariate and contrast analyses were performed in addition to multivariate analyses including all four microbial variables, and denominated as soil microbial variables (SMV). In general, microbial parameters and SMV were not affected by the transgene, type of herbicide or weed management. Differences were, rather, related to site, cropping season and cultivar.