Although bilateral animals appear to have left-right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. Here, we showed that Drosophila Jun N-terminal kinase (D-JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a D-JNK phosphatase, showed random laterality of the AMG. Directional LR looping of the AMG required D-JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of D-JNK signaling was required for the LR asymmetric looping. We also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) was regulated by D-JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augmented D-JNK signaling in this process. Our results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates.