Grassland ecosystems are influenced by natural and anthropogenic factors, of which soil, predominance of agriculturally valuable perennials, climatic conditions and management practices are the most important ones. The data on earthworms in grasslands are still scarce despite their importance for ecosystem functioning. Experiments were carried out at the Vėžaičiai Branch of Lithuanian Research Centre for Agriculture and Forestry during the period 2014-2016 on plain and hilly relief. The objective of this study was to investigate changes of the earthworm population in different grassland ecosystems under natural and anthropogenic conditions. The soil of the experimental sites was Bathygleyic Dystric Glossic Retisol. On the plain, where the soil acidity (pH) of the experimental site was 3.9 and 5.0, four mixtures of legumes and grasses were cultivated. The four mixtures were composed of one legume variety and two grass varieties -timothy (Phleum pratense L.) 35% and meadow grass (Poa pratensis L.) 15%, 50% of each: 1) red clover (Trifolium pratense L.), 2) white clover (Trifolium repens L.), 3) hybrid clover (Trifolium hybridum L.) and 4) alfalfa (Medicago sativa L.). On hilly relief, samples were collected from the soil of the 1) temporary: alfalfa (Medicago sativa L.) 50% + timothy (Phleum pratense L.) 35% + meadow grass (Poa pratensis L.) 15%, and 2) permanent (established in 1983): timothy (Phleum pratense L.) 20%, red fescue (Festuca rubra L.) 20%, meadow grass (Poa pratensis L.) 20%, white clover (Trifolium repens L.) 20%, common bird's-foot trefoil (Lotus corniculatus L.) 20%, grasslands grown on the slopes of the southern exposition, with a slope of 14-16° and the northern exposition, with a slope of 11-13°. The number and biomass of earthworms depended on the soil acidity, hill exposition and grassland age (permanent grassland or temporary grassland in a crop rotation). In temporary grasslands on the plain, the greatest number and biomass of earthworms were determined in the soil with an acidity of 5.0. On hilly relief with different soil moisture conditions, where the soil acidity was ≥5.7, the greatest number and biomass of earthworms were determined in the soil of the northern exposition, irrespective of the sward age.