Left-right (LR) asymmetry is a conserved characteristic of the brain in various animals and is related to its higher-order functions. The Drosophila brain has an LR asymmetric structure known as an asymmetrical body (AB). LR asymmetric neurite remodeling lateralizes the AB, and ecdysone signaling determines LR specificity. However, the mechanisms underlying LR specificity remain unclear. We found that the Slit/Dreadlocks/Roundabout/p21-activated kinase (Pak) signaling axis determines the LR polarity of the AB downstream of ecdysone signaling in the type II neuroblast lineage before LR asymmetric neurite remodeling. In Drosophila, the intrinsic chirality of cells (cell chirality) defines the LR asymmetry of various non-neuronal organs. We suggested that neurons derived from type II neuroblasts exhibit cell chirality, which is established through Pak and ecdysone signaling and determines the LR polarity of the AB. As cell chirality is broadly observed in eukaryotes, our study reveals a novel mechanism underlying LR asymmetry of the brain.