The extent to which long−term no tillage alters the quantity and sources of soil organic matter (SOM) in the soil profiles remains elusive. Here, using a two-biomarker approach (i.e., lignin phenols and amino sugars), we quantified the origins of SOM, that is, plant lignin and microbial necromass, under three tillage practices in different soil layers, including moldboard plowing (MP), subsoiling (SS), and no tillage (NT) from a 20−yr trial in Northern China. The results showed that SS (cf. MP) enhanced soil organic C storage by 20% in the surface of 0–10 cm layer rather than the deeper profile (> 10 cm). In the surface soils, tillage affected plant−derived lignin phenol concentrations, NT > SS > MP, but not microbial necromass C (MNC), likely because of stubble enrichment in the topsoil. Instead, NT (cf. MP) increased the fungal necromass C and MNC concentrations by 60% and 14% in the sublayer (30–40 cm). Furthermore, NT enhanced microbial-derived C storage (mainly fungal−derived C) compared to MP in this deeper layer. The increased contribution and distribution of microbially derived C with soil depth refined our mechanistic understanding of SOM formation after tillage transition. We conclude that long−term no tillage can alter SOM origins rather than storage, and thus, the potential persistence is due to accumulated microbial necromass in the sublayer.