Background: Tertiary hyperparathyroidism (THPT) is very common in hemodialysis patients with secondary hyperparathyroidism. However, a medical treatment is not indicated for THPT. Purpose: To investigate the feasibility, safety and efficacy of microwave ablation (MWA) in treating THPT. Materials and methods: Twenty-three patients with THPT were enrolled and treated with MWA. Clinical characteristics, serum levels of intact parathyroid hormone (iPTH), calcium, phosphorus and alkaline phosphatase (ALP), as well as treatment outcomes, were evaluated pre-and post-MWA. All patients were followed for >36 months for all assessable clinical data. Results: All patients successfully completed MWA. The mean follow-up was 47.0 ± 8.4 months. Immediately 1-day post-MWA, iPTH, calcium, phosphorus and ALP levels significantly decreased (all p < 0.001). During the long-term follow-up, iPTH levels increased gradually until 24 months and then remained at stable levels. After MWA, serum calcium reached stable levels at 24 months, while phosphorus and ALP reached stable levels at 6 months, and the levels were in the normal range or slightly higher than the upper normal limit. No obvious blood flow signals or significant recurrence was observed in the surgical nodules during follow-up. In the last follow-up, all nodules were persistent, but their maximum diameter and average volume were significantly lower after MWA (both p < 0.001), with an average reduction of 75.9 ± 11.3%. All patients had no major complications during MWA and follow-up. Conclusions: MWA is feasible, safe, effective and minimally invasive in treating THPT. Thus, MWA can be a nonsurgical alternative for treating THPT patients who are ineligible for surgery.