Long-term manure application has the potential to alleviate soil acidification, and increase carbon sequestration and nutrient availability, thus improving cropland fertility. However, the mechanisms behind greenhouse gas N₂O emissions from acidic soil mediated by long-term manure application remain poorly understood. Herein, we investigated N₂O emission and its linkage with gross N mineralization and nitrification rates, as well as nitrifying and denitrifying microbes in an acidic upland soil subjected to 36-year fertilization treatments, including an unfertilized control (CK), inorganic fertilizer (F), 2× rate of inorganic fertilizer (2F), manure (M), and the combination of inorganic fertilizer and manure (FM) treatments. Compared to the CK treatment (1.34 μg N kg^–1 d^–1), fertilization strongly increased N₂O emissions by 34-fold on average, with more pronounced increases in the manure-amendment (10.6–169 μg N kg^–1 d^–1) than those in the inorganic fertilizer treatments (3.26–5.51 μg N kg^–1 d^–1). The manure amendment-stimulated N₂O emissions were highly associated with increased soil pH, mean weight diameter of soil aggregates, substrate availability (e.g., particulate organic carbon, NO₃- and available phosphorus), gross N mineralization rates, denitrifier abundances and the (nirK+nirS)/nosZ ratio. These findings suggest that the increased N₂O emissions primarily resulted from alleviated acidification, increased substrate availability and improved soil structure, thus enhancing microbial N mineralization and favoring N₂O-producing denitrifiers over N₂O consumers. Moreover, ammonia-oxidizing bacteria (AOB) rather than ammonia-oxidizing archaea (AOA) positively correlated with soil NO₃^- concentration and N₂O emissions, indicating that nitrification indirectly contributed to N₂O production by supplying NO₃^- for denitrification. Collectively, manure amendment potentially stimulates N₂O emissions, primarily resulting from alleviated soil acidification and increased substrate availability, thus enhancing N mineralization and denitrifier-mediated N₂O production. Our findings suggest that consideration should be given to the greenhouse gas budgets of agricultural ecosystems when applying manure for managing the pH and fertility of acidic soils.