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Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction of light between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones, but also an opposite response in neighboring no-light growth cones of the same neuron. Finally, this tool was used to reorient growth cones towards or away from light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.Movie S1Movie S1Actin dynamics of DRG growth cones before blue light exposure.Pseudocolored Lifeact movies of DRG growth cones expressing CIB1 (left), iPlex‐Opto (middle), or iTrk‐Opto (right). The movie is created from images taken every 2 seconds for a total of 10 minutes before blue light exposure. The movie is shown at 30 fps.Movie S2Movie S2Changes in actin dynamics of DRG growth cones expressing CIB1, iPlex‐Opto and iTrk‐Opto in the presence of blue light.Pseudocolored Lifeact movies of DRG neurons expressing CIB1 (left), iPlex‐Opto (middle), or iTrk‐Opto (right). Growth cones were imaged for Lifeact while simultaneously exposed to blue light every 2 seconds for a total of 10 minutes. iPlex‐Opto growth cones undergo transition to collapsed while CIB1 and iTrk‐Opto remain dynamic. The movie is shown at 30 fps.Movie S3Movie S3Actin dynamics in DRG axons prior to blue light exposure.Pseudocolored Lifeact movies of DRG axons expressing either CIB1 (left), iPlex‐Opto (middle), or iTrk‐Opto (right). The movie is from created from images taken every 5 seconds for a total of 5 minutes, and shown at 30 fps.Movie S4Movie S4Changes in actin dynamics after localized blue light exposure.Pseudocolored Lifeact movies of DRG axons expressing CIB1 (left), iPlex‐Opto (middle) or iTrk‐Opto (right). The movie is taken right after exposed to localized blue light every 5 seconds for a total of 5 minutes and shown at 30 fps. Local activation of iTrk‐Opto induced the formation of lamellipodia at the sight of activation.