| Summary: | Alfalfa (<i>Medicago sativa</i> L.) and corn (<i>Zea mays</i> L.) are common forage plants for feeding livestock, and their effects on soil bacterial organisms have been extensively studied. However, there is little comprehensive research on soil bacterial organisms and their function in a long-term alfalfa monocropping system after corn insertion and fertilization. The effects of alfalfa–corn rotation (AF: alfalfa monocropping, RA: alfalfa and corn rotation) and nitrogen fertilization (RA0 and RA15) were investigated in a field experiment. The results showed that fertilization significantly increased the aboveground biomass (AGB) and soil nitrate nitrogen, and corn insertion significantly decreased the nitrate nitrogen (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mrow><mi>NO</mi></mrow><mn>3</mn><mo>−</mo></msubsup></mrow></semantics></math></inline-formula>-N) (<i>p</i> < 0.05). In addition, the bacterial community structure among the treatments was significantly changed by the rotation and fertilization. The rotational system of RA significantly increased the soil bacterial diversity compared with AF (<i>p</i> < 0.01), and most of the soil bacterial communities were of the phyla Actinobacterota and Proteobacteria. The RA system had a lower relative abundance of Actinobacterota than the AF system. The bacterial function prediction found that the soil carbon and nitrogen cycle processes in RA were more active than those in AF. The RDA analysis revealed that <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mrow><mi>NO</mi></mrow><mn>3</mn><mo>−</mo></msubsup></mrow></semantics></math></inline-formula>-N and pH were the main environmental factors affecting the bacterial community structure in the RA system.
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