No-tillage (NT) systems have arisen as a promising alternative to traditional rice cultivation in flooded soils, such as pre-germinated (PG) and conventional tillage (CT) systems. However, how NT affects soil properties and grain yield is not completely understood, especially over the long term. The objective of this study was to evaluate the long-term impact of three tillage systems (CT, NT and PG) on soil chemical, physical and biological attributes and the influence of these attributes on rice yield over time in a lowland soil of the Brazilian subtropical region. The rice grain yield was evaluated for 16 growing seasons, and the soil properties were evaluated from 17 to 21 years after the beginning of the experiment. After 14 growing seasons, rice yield was greater (3.4%) under the NT system compared to under the CT system. The increase in rice yield over time resulted in 67% greater soil organic matter (OM) content, exchangeable Ca and Mg and consequently cation exchange capacity (CEC) under the NT system compared to the CT and PG systems. In addition, soil bulk density and microbial C and N were 15%, 40% and 75% greater, respectively, under the NT system than under the CT system in the surface soil layer. The lack of soil disturbance associated with the high input of crop residues and flooding soil conditions favored the chemical, physical and biological soil properties through soil OM content, improving rice yields after 14 yr of NT adoption in relation to CT adoption.