Context. The effects of no-tillage (NT) on soil organic C (SOC) and rice yield in lowland soils are poorly understood in subtropical ecosystems. Aims. In a long-term (24 years) field experiment, we assessed the effect of NT on SOC stocks in labile (>53 μm, particulate) and stable (<53 μm, mineral associated) fractions of soil organic matter (SOM), grain yield, between-season stability, and gross margin compared to conventional tillage (CT) and pre-germinated tillage (PG) systems in a lowland Gleysol in southern Brazil. Methods. Soil from eight different layers down to 40 cm under each tillage system was sampled in a field experiment, and additional soil samples were obtained from an adjacent area under native grassland (NG) as reference for SOC stocks. Key results. While the PG and CT systems maintained similar SOC stocks as the NG soil, NT increased SOC stocks at an annual rate of 0.41 Mg ha−1 in relation to the traditional CT soil. Rice grain yield increased twice over the 24-year period, amounting to 12 Mg ha−1 in the last crop season. Despite lower yield in NT system in most crop seasons, a slight effect on yield stability and profitability (<10%) in favour of NT, compared with CT and PG systems, was observed. Conclusions. Based on our findings, NT promotes C accumulation in subtropical paddy rice soils. Implications. There is a beneficial effect of NT on rice yield stability and profitability in the long term.