Aims <p>The objective of this study was to evaluate the effect of different mixed grass-legume pastures compared to single grass cultivation as the second crop of an integrated crop livestock system (ICLS) in Brazilian Cerrado on (i) soil organic carbon and nitrogen pools, (ii) microbial biomass, enzyme and microbial activity of the soil, (iii) soil health, and (iv) soybean grain yield in succession.</p> Methods <p>In this experiment, the soybean was cultivated in the summer season, with the subsequent treatments with two grasses (<i>Urochloa ruziziensis</i> or <i>Urochloa brizantha</i>), single or intercropped with Cowpea (<i>Vigna unguiculata</i>) or Pigeon pea (<i>Cajanus cajan</i>), and soil collections were carried out 2&#xa0;years after the implementation of the experiment, which was in 2015.</p> Results <p>Regardless of the grass species, Cowpea intercropping yielded 28% more soybeans than single-cropped grasses. The intercropping effects on soybean yield were directly related to improvements in soil biological and biochemical properties since there was a higher microbial biomass and activity, and enzymatic activity in the soil. In addition to the higher basal respiration and metabolic quotient (<i>q</i>CO₂), the lower microbial quotient (<i>q</i>MIC) indicates a microbial stress condition in grass monoculture compared to mixed grass-legume pastures.</p> Conclusions <p>The mixed grass-legume pastures are effective strategies to increase C and N stocks in different soil pools under integrated crop-livestock systems, reflecting increases in soybean grain yield. Grass-legume intercropping in the pasture phase of ICLS is an additional tool to maximize soil health improvements and soybean yields in the short term.</p> Graphical abstract <p></p>