In this study, our investigated hypothesis was that different pre-crops would have different effects on earthworm activity and soil CO2 emissions. We also hypothesized that a pre-crop clover\u2013timothy mixture would perform best in terms of increasing the share of organic carbon in soil and, in this way, contribute to improving the sustainability of agroecosystems. The aim of this study was to explore the residual effects of using a 50-year-term of three different crop rotations and a continuous bare fallow period on soil CO2 emissions by investigating the soil earthworm populations, soil agrochemical properties, and winter wheat yields. A field experiment was carried out from 2016 to 2017 at Vytautas Magnus University in Lithuania (54\u00b053\u2032 N, 23\u00b050\u2032 E). The experiment was conducted in crop stands of winter wheat cv. \u2018Skagen\u2019, which were sown in three crop rotations with different pre-crops and a continuous bare fallow period. The pre-crop used for winter wheat in the cereal crop rotation (CE) was a vetch and oat mixture for green forage, LEG-CER; the pre-crop used for winter wheat in the field with row crops (FWR) crop rotation was black fallow, FAL-CER; the pre-crop used for winter wheat in the Norfolk (NOR) crop rotation was a clover\u2013timothy mixture, GRS-CER; and finally, continuous bare fallow, FAL-CONTROL, was used as well. The highest soil CO2 emission intensity was determined after the pre-crops that left a large amount of plant residues (clover and timothy mixture) in the soil. Plant residues remaining after the pre-crop had the greatest effect on the number of earthworms in the soil after the harvesting of winter wheat. Winter wheat had the best yield when grown in grass and legume sequences. Crop rotation sequences that included perennial grasses accumulated higher contents of total nitrogen and organic carbon. The best values for the productivity indicators of wheat were obtained when it was grown after a fallow crop fertilized with cattle manure. An appropriate crop rotation that promotes the steady long-term contribution of organic matter and increases the content of organic carbon in the soil will have a positive effect on the agrochemical, biological, and physical properties of soil and agroecosystem sustainability; moreover, these effects cannot be achieved by technological means alone.