Ensifer adhaerens, a microorganism recognized for its capacity to synthesize vitamin B12 (VB12), has garnered significant attention in recent years. Nonetheless, its practical application has been limited by low production yields. Atmospheric and room-temperature plasma (ARTP) mutagenesis was utilized to improve VB12 production and examine the associated mechanisms. Three high-yielding mutant strains─BCA-24, BCB-14, and BCC-27─were isolated through multiple rounds of mutagenesis. The VB12 titer of the highly productive mutant strain, BCA-24, rose significantly from 65.64 mg/L to 104.54 mg/L. Genome resequencing identified 14 mutated genes, of which seven (atpA, gntR, fusA, cobQ, ribD, cirA, and UP) were functionally validated through overexpression in wild-type strains and found to positively influence VB12 biosynthesis. Notably, coexpression of the cobQ and UP mutant genes in strain BCA-24 resulted in a VB12 titer of 163.68 mg/L. Transcriptomic analysis indicated that critical pathways related to energy metabolism, S-adenosylmethionine (SAM), 5-aminolevulinic acid (5-ALA), and riboflavin synthesis were significantly upregulated in BCA-24 relative to the wild type. A multiomics approach clarified the mechanisms through which these mutations increase VB12 production, including enhanced transcription and translation, optimized energy supply, and improved product efflux. The identification of novel candidate genes in Ensifer adhaerens, which have not been previously studied, provides valuable resources for future genetic engineering aimed at enhancing VB12 production efficiency. This study offers practical improvements in microbial VB12 production while also delivering essential insights into the genetic and metabolic regulation of this important biosynthetic pathway.