The pathogenesis of necrotic enteritis (NE) involves complex gene regulation at both the bacterial cell and host tissue levels, yet many aspects remain incompletely understood. This study aims to compare the differential transcriptome of the netB-positive Clostridium perfringens strain, CP54, before and after infection. Differentially expressed genes and pathways were also examined in jejunal tissues from CP54-induced and CP54-Eimeria coinfected NE models to identify potential targets for mitigating NE. Forty-one-day-old Cobb straight-run broilers were assigned to four groups: CP and Eimeria coinfection group (EimCP), Eimeria infection group (Eim), CP54 infection group (CP), and untreated control (CTL). Subclinical and severe NE models were established by oral infection with CP54 alone and CP54-Eimeria coinfection, respectively. Three post-infection CP54 strains (CPd1, CPd2, and CPd3) were isolated from necrotic jejunal mucosa in the EimCP group and analyzed alongside pre-infection CP54 using 4-plex bacterial RNA sequencing (RNA-seq). Jejunal tissues were collected and analyzed for differentially expressed genes between groups via tissue RNA-seq. The results showed that post-infection CP54 strains exhibited unique gene regulation patterns associated with environmental adaptation, including upregulation of oxidation-reduction processes, oxidoreductase activity, and downregulation of pyrimidine metabolism. However, no differential expressed virulence genes, including cpa, colA, lepB, luxS, nanI, netB, and cpb2, were identified between the pre- and post-infection CP54 cells. In host tissues, the analysis revealed significant activation of cytokine-cytokine receptor interactions and Toll-like receptor pathways that contribute to inflammatory responses. Upregulating IL8, IL12B, and INHBA played a key role in activating these pathways. Additionally, NE-infected jejunal tissues displayed suppressed PPAR pathway activity and increased p53 signaling. These changes suggest a significant role for apoptosis, immune regulation, and lipid metabolism in the progression of the disease. In summary, this study identifies key genes and transcripts associated with NE at both the bacterial and host levels, offering perspectives on the pathways driving disease progression and host-pathogen interactions. These findings provide crucial insights for developing effective prevention and control strategies, ultimately reducing NE risks and associated losses in the broiler industry.