WRKY transcription factors play essential roles in mediating various stress responses in plants. Although numbers of researches have investigated the functional mechanisms of WRKY TFs, relative to the research progress in model plants, rather limited numbers of WRKY TFs have been functionally characterized in cotton. In this study, we isolated and characterized GhWRKY44, a group I WRKY gene from cotton (Gossypium hirsutum). Subcellular localization indicated that GhWRKY44 was localized to the nucleus. Additionally, a group of cis-acting elements associated with the response to environmental stresses were predicted in the promoter. The expression of GhWRKY44 can be induced by pathogen injection, abiotic stresses and diverse signaling molecules. Furthermore, the overexpression of GhWRKY44 in N. benthamiana exhibited enhanced resistance to bacterial pathogen R. solanacearum and fungal pathogen R. solani compared with wild-type plants. Importantly, several defense-related genes were induced in the transgenic plants, including PR-1, PR-2, PR-5 and NPR1 for SA signaling, and PR-4 for JA signaling. The transgenic plants also exhibited lower levels of ROS (H2O2 and O-2 (-)) accumulation than wild-type plants following pathogen infection. Taken together, these results suggest that GhWRKY44 positively regulates pathogen induced plant disease resistance, and these findings will expand our knowledge on the functions of WRKY TFs in multiple signal transduction pathways of disease resistance.