The peduncles of Tripsacum laxum Nash were used as explants to induce axillary shoots. Multiple shoots were proliferated on Murashige and Skoog (MS) medium to establish, for the first time, efficient shoot proliferation and plant in vitro regeneration systems. Optimal shoot proliferation medium was MS with 3.0 mg/L 6-benzyladenine (BA) and 0.2 mg/L α-naphthaleneacetic acid (NAA), resulting in a shoot proliferation coefficient of 11.0 within 45 d. Optimal rooting medium was MS with 0.1 mg/L NAA and/or 0.1 mg/L indole-3-butyric acid (IBA), inducing 100% root formation from shoots within 30 d. When young roots, leaf sheaths and shoot bases were used as explants, MS medium with 1.0 mg/L thidiazuron (TDZ) and 0.2 mg/L BA induced most shoots, with the least callus. Shoot bases induced beige-white callus and shoots directly on MS medium with 1.0 mg/L TDZ and 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), while leaf sheaths induced beige-white callus and shoots directly on MS medium with 1.0 mg/L TDZ and 0.2 mg/L BA. Rooted plantlets showed 99.3% survival when transplanted into a substrate of vermiculite: peat soil (1:3, v/v).