Glycolate oxidase (GOX) plays an important role in the regulation of photorespiration and photosynthesis in plants. However, as one of the main enzymes to produce the second messenger hydrogen peroxide (H2O2), its functions in response to pathogens are still poorly understood. In this study, we carried out genome-wide identification, and 14 GOX genes were identified in Gossypium hirsutum. These GOX genes are located on 10 chromosomes and divided into hydroxyacid-oxidases (HAOX) and GOX groups. After infection with Verticillium dahliae Kleb., six GOX gene expression levels were changed. Moreover, H2O2, salicylic acid (SA), and the content and activity of GOX increased in cotton. GhHAOX2-D-silenced plants showed more wilting than control plants after infection with V. dahliae. Additionally, H2O2 accumulation and SA content were reduced in GhHAOX2-D-silenced plants. The expression levels of GhPAL, GhPAD4, and GhPR1 and the lignin content of the silenced plants were significantly lower than those of the control plants. These results indicate that GhHAOX2-D is a positive regulator of Verticillium wilt tolerance in cotton and may promote H2O2 accumulation via the synergistic effects of GOX genes and SA. Collectively, GOX genes play an important role in cotton resistance to Verticillium wilt.