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Poultry litter biochar (PLB) is a value-adding soil amendment and an economically sustainable approach that is used to enhance food safety and reduce environmental harm. Poultry litter biochar has promising potential but has been under-examined in regards to carbon (C) sequestration in relation to its type and application rate. The objective of this study was to investigate the effectiveness of PLB in enhancing the C sequestration of acid soils through a short-term incubation experiment. The soil was amended with different materials: PLB (1%, 5%, and 10%) and a control (non-amended). The results indicated that PLB application increased soil C mineralization relative to the control (19–1562%), it significantly increased with an increasing application rate (e.g., increased addition 29, 99, and 172% for 1, 5, and 10% of 400 °C PLB), and the soil C mineralization and applied carbon mineralized (ACM) significantly decreased with temperature (e.g., the cumulative C pool ranges of ACM with 1% PLB, added at pyrolysis temperatures of 200, 300, 400, 500, and 600 °C, were 42.0, 34.4, 19.6, 6.16, and 4.04%, respectively). To assist sustainable soil management and to aid the achievement of multiple sustainable development goals (SDGs), as well as to maximize the benefits of PLB applications and minimize the potential environmental risk, it is suggested that application of PLB, pyrolyzed within 400–600 °C at a rate between 1% to 5%, should be adopted in acidic soils in Taiwan. [ABSTRACT FROM AUTHOR]