Ca2+ signals are known to be important regulators of neurite outgrowth and steering. Here we show that inhibiting Ca2+ influx through stretch-activated channels using various compounds, including a highly specific peptide isolated from Grammostola spatulata spider venom (GsMTx4), strongly accelerates the rate of neurite extension on diverse substrata and within the intact spinal cord. Consistent with the presence of stretch-activated channels, we show that Ca2+ influx is triggered by hypotonic solutions, which can be partially blocked by GsMTx4. Finally, chelating local, but not global, Ca2+ signals prevents the acceleration that is normally produced by GsMTx4. Blocking Ca2+ influx through other channel types has little or opposite effects, but release from intracellular stores is required for maximal acceleration. Together, our data suggest that Ca2+ functions at distinct microdomains in growth cones, with influx through mechanosensitive channels acting to inhibit outgrowth in opposition to influx through other plasma membrane channels and release from stores.