Neonatal Meningitis-causing (NMEC) is the leading cause of neonatal meningitis and exhibits remarkable adaptability to diverse host environments. Understanding its transcriptional responses across different host niches is crucial for deciphering pathogenesis and identifying potential therapeutic targets. We performed a comparative transcriptomic analysis of NMEC RS218, the prototype strain of NMEC, under four distinct host-mimicking conditions: colonic fluid (CF), serum (S), human brain endothelial cells (HBECs), and cerebrospinal fluid (CSF). Differential gene expression analysis was conducted to assess metabolic shifts, virulence factor regulation, and niche-specific adaptation strategies, in which RS218 demonstrated niche-specific transcriptional reprogramming. In CF, genes associated with biofilm formation, motility, efflux pumps, and cell division regulation were upregulated, aiding gut colonization. The serum environment triggered the expression of siderophore-mediated iron acquisition, enterobactin biosynthesis, and heme utilization genes, facilitating immune evasion and bacterial persistence. In HBECs, NMEC upregulated genes linked to nucleoside metabolism, membrane remodeling, pilus organization, and blood-brain barrier (BBB) traversal. In CSF, genes related to oxidative stress resistance, chemotaxis, DNA repair, biofilm formation, and amino acid biosynthesis were enriched, reflecting NMEC's adaptive mechanisms for survival under nutrient-depleted conditions. Energy-intensive pathways were consistently downregulated across all niches, highlighting the need for an energy conservation strategy. This study provides novel insights into NMEC's adaptive strategies across different host environments, emphasizing its metabolic flexibility, virulence regulation, and immune evasion mechanisms, offering potential targets for therapeutic intervention.