Background: Hashimoto's Thyroiditis a common disease that affects the thyroid gland. Recent studies on Hashimoto's Thyroiditis indicated the ability of miRNA to silence messenger RNA and regulate post-transcriptional processes. Objective: The aim of the current study was to find the relationship between microRNA 125a-5p and the production of antibodies thyroid peroxidase (Anti-TPO). Patients and methods: The present study enrolled 125 patients, whom 25 were males and 100 females; the ratio of males to females was 1:4. Blood samples were collected from the participants from the Specialized Center for Endocrinology and Diabetes, Rusafa, Baghdad (Iraqi Ministry of Health). Hashimoto's patients were diagnosed using clinical methods, ultrasound, and serological testing. Also, anti-TPO were detected using the ELASI technique. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect microRNA125a-5p levels in 40 samples from patients with Hashimoto's disease and compare them with 10 healthy individuals. Results: There were high levels of Anti-TPO in patients with Hashimoto's disease compared with the healthy individuals, with a statistically significant difference (P ≤0.01). The study also showed a decrease in the levels of miRNA 125a-5p in patients with Hashimoto's disease compared with the healthy individuals, with a statistically significant difference (P ≤0.01). Conclusion:The study showed a decreased miRNA-125a-5p expression that was found to be inversely associated with the low level for gene expect. This could be related to many factors as patients might be taking medication, population differentiation, races, miRNA, and drug interaction.