The rhizobacterium Pseudomonas protegens H78 biosynthesizes a number of antibiotic compounds, including pyoluteorin, 2,4-diacetylphloroglucinol, and pyrrolnitrin. Here, we investigated the global regulatory function of the nitrogen metabolism-related sigma factor RpoN in P. protegens H78 through RNA-seq and phenotypic analysis. During the mid- to late-log growth phase, transcriptomic profiling revealed that 562 genes were significantly upregulated, and 502 genes were downregulated by at least twofold at the RNA level in the rpoN deletion mutant in comparison with the wild-type strain H78. With respect to antibiotics, Plt biosynthesis and the expression of its operon were positively regulated, while Prn biosynthesis and the expression of its operon were negatively regulated by RpoN. RpoN is responsible for the global activation of operons involved in flagellar biogenesis and assembly, biofilm formation, and bacterial mobility. In contrast, RpoN was shown to negatively control a number of secretion system operons including one type VI secretion system operon (H1-T6SS), two pilus biogenesis operons (Flp/Tad-T4b pili and Csu-T1 pili), and one polysaccharide biosynthetic operon (psl). In addition, two operons that are involved in mannitol and inositol utilization are under the positive regulation of RpoN. Consistent with this result, the ability of H78 to utilize mannitol or inositol as a sole carbon source is positively influenced by RpoN. Taken together, the RpoN-mediated global regulation is mainly involved in flagellar biogenesis and assembly, bacterial mobility, biofilm formation, antibiotic biosynthesis, secretion systems, and carbon utilization in P. protegens H78.