The incidence of thyroid nodules has been increasing worldwide; however, there are currently no feasible and robust methods to differentiate malignant thyroid nodules from benign thyroid nodules. The present study aimed to establish a practical method to determine the malignancy of thyroid nodules. Reverse transcription‑quantitative PCR and western blot analyses were performed to compare the levels of long non‑coding RNA nuclear enriched abundant transcript 1 (NEAT1), microRNA (miR)‑9, miR‑124, PTEN and programmed cell death protein 6 (PDCD6) in the peripheral blood and thyroid tissue samples between patients with malignant and benign thyroid nodules. Additionally, a regulatory relationship between NEAT1, miR‑124, miR‑9, PTEN and PDCD6 was established in the present study. The diagnostic value of NEAT1, miR‑124 and miR‑9 was determined using a ROC analysis. The expression levels of NEAT1, PTEN and PDCD6 in peripheral blood and thyroid tissue samples collected from the benign group were higher compared with those in the malignant group, whereas the expression levels of miR‑124 and miR‑9 were lower in the benign group. In the peripheral blood, NEAT1 expression exhibited an area under the curve (AUC) value of 0.8546, whereas miR‑124 and miR‑9 expression had AUC values of 0.7657 and 0.7019, respectively. In the thyroid tissue, NEAT1, miR‑124, and miR‑9 had AUC values of 0.9304, 0.8221 and 0.7757, respectively. Additionally, miR‑9 and miR‑124 expression levels in BCPaP and SW579 cells was decreased after transfection with a NEAT1 expression vector compared with those in cells transfected with the control vector, whereas the expression of PTEN and PDCD6 was upregulated. By contrast, transfection with short hairpin RNA targeting NEAT1 notably increased the expression of miR‑9 and miR‑124 while downregulating the expression of PTEN and PDCD6 compared with that in the control cells. In conclusion, the results of the present study demonstrated that the dysregulation of NEAT1 expression may be used to differentiate benign and malignant thyroid nodules.