To evaluate safety and efficacy of one- vs. two-session radiofrequency ablation (RFA) of parathyroid hyperplasia for patients with secondary hyperparathyroidism (SHPT) and to compare the outcome of both methods on hypocalcemia. Patients with secondary hyperparathyroidism underwent ultrasound guided RFA of parathyroid hyperplasia. Patients were alternately assigned to either group 1 (n = 28) with RFA of all 4 glands in one session or group 2 (n = 28) with RFA of 2 glands in a first session and other 2 glands in a second session. Serum parathyroid hormone (PTH), calcium, phosphorus and alkaline phosphatase (ALP) values were measured at a series of time points after RFA. RFA parameters, including operation duration and ablation time and hospitalization length and cost, were compared between the two groups. Mean PTH decreased in group 1 from 1865.18 ± 828.93 pg/ml to 145.72 ± 119.27 pg/ml at 1 day after RFA and in group 2 from 2256.64 ± 1021.72 pg/ml to 1388.13 ± 890.15 pg/ml at 1 day after first RFA and to 137.26 ± 107.12 pg/ml at 1 day after second RFA. Group 1's calcium level decreased to 1.79 ± 0.31 mmol/L at day 1 after RFA and group 2 decreased to 1.89 ± 0.26 mmol/L at day 1 after second session RFA (P < 0.05). Multivariate analysis showed that hypocalcemia was related to serum ALP. Patients with ALP ≥ 566 U/L had lower calcium compared to patients with ALP < 566 U/L up to a month after RFA (P < 0.05). Group 1's RFA time and hospitalization were shorter and had lower cost compared with Group 2. US-guided RFA of parathyroid hyperplasia is a safe and effective method for treating secondary hyperparathyroidism. Single-session RFA was more cost-effective and resulted in a shorter hospital stay compared to two sessions. However, patients with two-session RFA had less hypocalcemia, especially those with high ALP.