No studies have investigated whether the discontinuation of ethambutol (EMB) based on susceptibility to isoniazid and rifampin, as determined by the GenoType MTBDRplus assay, would be appropriate. We aimed to determine the feasibility of discontinuing EMB before the end of the intensive phase of treatment based on the results of the MTBDRplus assay in patients with pulmonary tuberculosis (PTB). This prospective, multicenter noninferiority randomized trial was conducted at 12 referral centers in South Korea in drug-susceptible PTB patients who initiated the standard four-drug regimen for PTB. Based on the results of the assay, EMB was discontinued in the MTBDRplus group after confirmation that the M. tuberculosis isolate was susceptible to isoniazid and rifampin. The time point for EMB discontinuation in the guideline group was determined using the results of the phenotypic drug susceptibility test based on the South Korean national tuberculosis guidelines. The primary outcome was treatment success. Secondary outcomes included the 1-year rates of recurrence and adverse events. Of 600 randomized patients, the treatment outcome analysis was performed for 493 patients (MTBDRplus group, 244 patients; guideline group, 249 patients). Treatment success rates were 93.9% (229/244) in the MTBDRplus group and 93.6% (233/249) in the guideline group and did not differ between groups (relative risk, 1.00; 95% confidence interval, 0.95 to 1.06). The 1-year recurrence rates in the two groups (0.9% and 0.5%, respectively) and adverse drug reactions did not differ between the groups. In conclusion, the early discontinuation of EMB based on the results of the MTBDRplus assay did not affect the treatment outcomes in patients with PTB. (This clinical trial has been registered with the Clinical Research Information Service of the Republic of Korea [https://cris.nih.go.kr/cris/] under registration no. KCT0000610.)