This prospective, randomized controlled trial evaluated the effect of neostigmine for intraoperative neuromonitoring (IONM) during thyroid surgery. Forty subjects undergoing thyroidectomy with IONM, randomized into neostigmine administration after tracheal intubation (Group N, n = 20) or control treatment with normal saline (Group C, n = 20), completed the trial. Electromyography amplitudes of the vagus nerve (V1) were recorded before thyroid dissection. The time from the initial V1 signal check to successful V1 stimulation was recorded. In Group N, all the patients had a successful V1 signal at the first check, whereas ten (50%) patients in Group C had a time delay between the initial V1 check and successful V1 (p < 0.001). The mean delay time among the delayed patients in Group C was 11.2 ± 1.4 min. The mean time from skin incision to successful V1 stimulation was significantly shorter in Group N than in Group C (15.4 ± 2.4 min vs. 19.9 ± 5.7 min, p = 0.003). In Groups N and C, the mean V1 amplitudes were 962.2 ± 434.5 μV vs. 802.3 ± 382.7 μV (p = 0.225), respectively, and the mean R1 amplitudes were 1240.0 ± 836.5 μV vs. 1023.4 ± 455.8 μV (p = 0.316), respectively. There was one bucking event in Group N. In conclusion, neostigmine administration immediately after tracheal intubation can be useful to reverse neuromuscular blockade for successful IONM in thyroid surgeries.