Nitrogen (N) fertilization and plastic film mulching (PFM) are two widely applied management practices for crop production. Both of them impact soil organic matter individually, but their interactive effects as well as the underlying mechanisms are unknown. Soils from a 28-year field experiment with maize monoculture under three levels of N fertilization (0, 135, and 270 kg N ha−1 yr−1) and with or without PFM were analyzed for soil organic C (SOC) content, total soil nitrogen (N), root biomass, enzyme activities, and SOC mineralization rates. After 28 years, N fertilization increased root biomass and consequently, SOC by 26% (averaged across the two fertilizer application rates) and total soil N by 25%. These increases, however, were only in soil with PFM, as PFM reduced N leaching and loss, as a result of a diurnal internal water cycle under the mulch. The SOC mineralization was slower with N fertilization, regardless of the PFM treatment. This trend was attributed to the 43% decrease of β-glucosidase activity (C cycle enzyme) and 51% drop of leucine aminopeptidase (N cycle) with N fertilization, as a result of a strong decrease in soil pH. In conclusion, root biomass acting as the main source of soil C, resulted in an increase of soil organic matter after 28 year of N fertilization only with PFM. © 2021 Elsevier B.V.