Objective To explore the expression and biological significance of Thyroid Transcription Factor-1 (NKX2-1) in thyroid cells of patients with toxic diffuse goiter.Methods Patients with toxic diffuse goiter treated in the Endocrinology and Thyroid Surgery Department of our hospital from January 2012 to January 2020 were selected as the study subjects. Thyroid tissues from 300 toxic diffuse goiter patients were collected as the observation group, and 100 healthy thyroid tissues were collected as the control group. Basic data of both groups were collected, and the expression levels of NKX2-1 and different signaling pathways proteins in both groups were assessed by qRT-PCR. Cell lines with silenced NKX2-1 and overexpressed NKX2-1 lentiviral vectors were constructed to compare cell proliferation and cloning ability under both conditions.Results In the observation group, the proportion of females was higher than in the control group (P <0.05). The titers of TRAb and TPOAb were both higher in the observation group than in the control group (P <0.05). The relative expression of NKX2-1 in the observation group was higher than in the control group (P <0.05). The cell proliferation rate and colony formation rate of the NKX2-1 overexpressing cell line were higher than those of the NKX2-1 silenced cell line (P <0.05). Comparing the absorbance values of NKX2-1 overexpressing and NKX2-1 silenced cell lines cultured for 1 day, 2 days, 4 days, 6 days, and 8 days, using repeated measures ANOVA, the results showed: (1) Comparison of absorbance values at different time points showed statistically significant differences (P <0.05); (2) The comparison of absorbance values between NKX2-1 overexpressing and silenced cell lines showed significant differences (P <0.05), with the NKX2-1 overexpressing group having higher absorbance values and better proliferative capacity; (3) The comparison of the trends in absorbance values between the two groups showed statistically significant differences (P <0.05).Conclusion NKX2-1 is significantly associated with the proliferation of thyroid cells in toxic diffuse goiter patients and can play a vital role in patient prognosis evaluation and treatment guidance.