Rationale: We recently reported strong bactericidal activity of the oxazolidinone PNU-100480 and its ability to increase the initial bactericidal effect of various combinations of first-line tuberculosis drugs and moxifloxacin in a murine model. Objectives and Methods: Therefore, we investigated whether the addition of PNU-100480 to the standard first-line regimen of rifampin, isoniazid and pyrazinamide could shorten the duration of treatment necessary to prevent relapse after treatment discontinuation. Measurements and Main Results: After 2 months of treatment mice receiving PNU-100480 in addition to the first-line regimen had lung CFU counts 2 orders of magnitude lower than control mice receiving the first-line regimen alone. Relapse rates after 4 months of treatment were 90%, 35% and 5% when PNU-100480 was added to the first-line regimen for 0, 2 and 4 months, respectively. When the total treatment duration was 3 months, relapse rates were 85% and 35-45% when mice received PNU-100480 for 2 and 3 months, respectively; all control mice remained culture positive at the time of treatment completion with 17-72 CFU per lung. Addition of linezolid to the first-line regimen had an antagonistic effect resulting in higher CFU counts and failure to render mice culture-negative in 4 months of treatment. Conclusions: Together with previous findings, these results confirm that PNU-100480, which is now in Phase I clinical testing, has sterilizing activity in the murine model and suggest that it may be capable of shortening treatment duration for drug-susceptible as well as drug-resistant tuberculosis in humans.