Nitric oxide (NO) is involved in diverse plant growth processes; however, little is known about pathways regulating NO levels in plants. In this study, we isolated a NO-overproducing mutant of tomato (Solanum lycopersicum) in which hyper-accumulation of NO, associated with increase in nitric oxide synthase (NOS)-like activity, caused diminished vegetative growth of plants and showed delayed flowering. The hyper-accumulation of NO caused drastic shortening of primary root (shr) in the seedlings, while the scavenging of NO restored root elongation in shr mutant. Inhibition of NOS-like activity reduced NO levels and stimulated root elongation in the shr mutant seedlings, while inhibition of nitrate reductase (NR) activity could not rescue shr phenotype. The stimulation of NO levels in shr mutant also conferred increased resistance to pathogen Pseudomonas syringae. Application of pharmacological inhibitors regulating ubiquitin–proteasome pathway reduced NO levels and NOS-like activity and stimulated shr root elongation. Our data indicate that a signaling pathway involving regulated protein degradation likely regulates NO synthesis in tomato.