The developmental role of the endocannabinoid system still remains to be fully understood. Here, we report thepresenceof a complete endocannabinoid system during zebrafish development andshowthat thegenes that code for enzymes that catalyze the anabolism and catabolism ( mgll and dagla ) of the endocannabinoid, 2‐AG (2‐arachidonoylglycerol), as well as 2‐AGmain receptor in the brain, cannabinoid receptor type 1, are coexpressed in defined regions of axonal growth. By using morpholino‐induced transient knockdown of the zebrafish Daglα homologandits pharmacologic rescue, we suggest that synthesis of 2‐AGis implicated inthe control of axon formationin themidbrain‐hindbrain region andthat animals that lack Dagl α display abnormal physiological behaviors intests that measure stereotypedmovement andmotion perception. Our results suggest that thewell‐established role for 2‐AG in axonal outgrowth has implications for the control of vision andmovement in zebrafish and, thus, is likely common to all vertebrates.—Martella, A., Sepe, R.M., Silvestri, C., Zang, J., Fasano, G., Carnevali, O., De Girolamo, P., Neuhauss, S.C. F., Sordino, P., Di Marzo, V. Important role of endocannabinoid signaling inthe development of functional vision and locomotion in zebrafish. FASEB J. 30, 4275–4288 (2016). www.fasebj.org