Calcium plays a key role during growth cone collapse. Recently, it has been proposed that low- and high-amplitude rises in [Ca(2+)](i) result in the activation of the neurite outgrowth inhibiting proteins calcineurin and calpain, respectively. However, it remains unknown if and how these mechanisms are modulated by specific guidance cues. Here we report that the inhibitory cue Semaphorin 5B induces growth cone collapse by promoting the influx of extracellular Ca(2+). The resulting rise in [Ca(2+)](i) is characterized by a low-amplitude increase followed by a marked secondary rise, suggesting the potential involvement of both calcineurin and calpain. In support of this, inhibition of either effector attenuated Sema5B-induced collapse, a result that was augmented by the simultaneous inhibition of both targets. Furthermore, we provide evidence that Sema5B induces calpain-mediated cleavage of calcineurin. We thus show for the first time that ligand-induced growth cone collapse can activate both calcineurin- and calpain-mediated pathways concurrently.