This research seeks to investigate how hypoxia inhibitors enhance iodine uptake in thyroid cancer cells. Clinical samples were gathered and assessed for hsa_circ_0023990, DNMT1, NIS, and their promoter methylation levels using RT-PCR, western blot, and methylation-specific PCR (MSP) techniques. The study involved examining the impact and mechanism of hsa_circ_0023990 on iodine uptake in differentiated thyroid carcinoma (DTC) cells through genetic manipulation. Luciferase reporter gene experiments were conducted to validate the interaction between hsa_circ_0023990, DNMT1, and miR-448. Xenograft tumors were established in nude mice for in vivo validation. The results showed that hsa_circ_0023990 was notably elevated in DTC and RAIR-DTC tissues. It was found that hsa_circ_0023990 could modulate NIS promoter methylation via the miR-448/DNMT1 signaling pathway, thereby influencing NIS expression. Hypoxia inhibitors effectively suppressed hsa_circ_0023990 expression in DTC cells, leading to increased NIS expression and enhanced iodine uptake. Subcutaneous transplantation experiments in animals further confirmed that hypoxia inhibitors could boost iodine absorption in tumor tissue and inhibit tumor growth through the hsa_circ_0023990/miR-448/DNMT1/NIS signaling axis. In conclusion, hypoxia inhibitors ameliorate iodine uptake dysfunction in thyroid cancer by acting on the hsa_circ_0023990/miR-448/DNMT1/NIS signaling pathway.