Background <p>Heroin addiction poses a significant threat to public health. Methadone maintenance therapy (MMT) is one of the effective treatments for heroin addiction, yet its molecular mechanisms remain largely unclear. This study aims to explore the potential key genes and underlying mechanisms involved in the therapeutic process of MMT for heroin addiction.</p> Methods <p>Samples were collected from 84 patients with heroin addiction and 84 patients undergoing MMT at the Fourth People’s Hospital of Urumqi. High-throughput transcriptome sequencing was used to identify differentially expressed genes (DEGs). Enrichment analysis and a protein-protein interaction (PPI) network were constructed for DEGs. RT-qPCR and Western Blot were used to validate the expression levels of key genes.</p> Results <p>Transcriptome sequencing identified 438 DEGs between the MMT group and the patients with heroin addiction group. Enrichment analysis suggested these genes were involved in biological processes such as cell cycle regulation and immune response. PPI network analysis indicated that PLK1 and IFIH1 occupied central positions in the network. PLK1 was involved in the FoxO signaling pathway, while IFIH1 was closely related to the RIG-I-like receptor signaling pathway. RT-qPCR and Western Blot results validated that the expression of PLK1 and IFIH1 was significantly downregulated in MMT patients compared to the patients with heroin addiction group, with statistical significance (<i>P</i> &lt; 0.05).</p> Conclusion <p>PLK1 and IFIH1 may play critical roles in MMT for heroin addiction, providing a scientific basis for further research into the molecular mechanisms and potential therapeutic targets of MMT.</p>