The Drosophila melanogaster bristle is a highly polarized cell that builds specialized cytoskeletal structures. Whereas actin is required for increasing bristle length, microtubules are essential for bristle axial growth. To identify new proteins involved in cytoskeleton organization during bristle development, we focused on identifying and characterizing the javelin (jv) locus. We found that in a jv mutant, the bristle tip is swollen and abnormal organization of bristle grooves is seen over the entire bristle. Using confocal and electron microscopy, we found that in jv mutant bristles, actin bundles do not form properly due to a loss of actin filaments within the bundle. We show that jv is an allele of the predicted CG32397 gene that encodes a protein with no homologs outside insects. Expression of the Jv protein fused to a green fluorescent protein (GFP) shows that the protein is colocalized with actin bundles in the bristle. Moreover, expression of Jv-GFP within the germ line led to the formation of ectopic actin bundles that surround the nucleus of nurse cells. Thus, we report that Jv is a novel actin-associated protein required for actin assembly during Drosophila bristle development.