The trends of enzyme activities and litter chemistry after abandonment of arable soil and succession of natural vegetation were studied in a deciduous forest zone (Moscow region, Russia). The Luvic Phaeozem chronosequence included an arable field, 3 fields with increasing abandonment periods (7, 11, 35 years), and the soil under natural forest as a reference site (never used for cropland). The activities of four hydrolytic enzymes (β-glucosidase, cellobiohydrolase, β-galactosidase, chitinase) in the topsoil (0–5 cm) were compared with chemical functional groups of plant litter identified by 13C NMR spectra. The total enzyme activity increased 5-fold during 35 years of postagricultural restoration of arable soil. The share of C-cycle enzymes with “narrower” substrate specificity (cellobiohydrolase and chitinase) increased during 35 years from 19% to 42% of the total enzyme pool. We explain this gain by the increase in the diversity and recalcitrance of organic compounds in the plant litter from cropland to natural forest. Aromaticity index of plant litter built up remarkably: from 0.11 in the young abandoned land to 0.14–0.15 in the 35-year abandoned land and the reference site. The share of enzymes with a “narrower” substrate specificity correlated closely with the portions of aryl C, O-aryl C, and carbonyl C functional groups in plant litter. Accordingly, the succession of natural vegetation on abandoned cropland leads to strong diversification of the plant litter composition and a corresponding production of soil enzymes with “narrower” substrate specificity. © 2020