Hemoglobins (Hbs) are evolutionarily conserved small globular proteins with characteristic 3-over-3 α-helical sandwich structure that is typically known as “globin fold”. Hbs have been found to be involved in diverse biological functions and the characteristic property of oxygen transportation is relatively a recent adaptation. Drosophila genome possesses three globin genes (glob1, glob2, and glob3) and it was previously reported that adequate expression of glob1 is required for various aspects of development, and also to regulate the cellular level of reactive oxygen species (ROS). The present study illustrates the explicit role of glob1 gene in Drosophila development. We demonstrate a dynamic expression pattern of glob1 in larval tissues which largely concentrate around F-actin rich structures and also co-precipitate. Reduced expression of glob1 leads to developmental abnormalities which appeared to be largely mediated by inappropriately formed F-actin based cytoskeletal structures. Our subsequent analysis in FLP/FRT mediated somatic clones establishes specific role of Drosophila glob1 in maintenance of the integrity of F-actin based cytoskeleton during development. For the first time, we report interaction between Glob1 and actin, and propose a novel role of glob1 in maintenance of F-actin based cytoskeleton in Drosophila.