Soil acidification due to the long-term application of nitrogenous fertilizers and the consequent impact on crop growth have been frequently reported. The effects of acidification on soil humus, charge, and microbial communities need to be studied. In this experiment, fertilizer drenching was used to simulate the effects of multiple years of fertilizer application on the black soil. The results showed that 25years of soil acidification treatment resulted in a decrease of 8.97% in the content of stable humus and led to a decrease of 58% and 51.18% in the humic acid (HA) content and degree of humification (PQ) value in stable humus, respectively. In addition, soil acidification leads to a significant decrease in total negative charge (CEC8.2) and variable negative charge (CECv), with both decreasing by 63.28% and 88.67%, respectively, at 25years. Acidification treatments affected both soil microbial community abundance and diversity, with a significant decrease in Acidobacteriota and Gemmatimonadota abundance and an increase in Bacteroidia abundance and Acidobacteriota abundance at 25years.