Wnt proteins are conserved axon guidance cues that control growth cone navigation. However, the intracellular signaling mechanisms that mediate growth cone turning in response to Wnts are unknown. We previously showed that Wnt–Frizzled signaling directs spinal cord commissural axons to turn anteriorly after midline crossing through an attractive mechanism. Here we show that atypical protein kinase C (aPKC), is required for Wnt-mediated attraction of commissural axons and proper anterior–posterior (A–P) pathfinding. A PKCζ pseudosubstrate, a specific blocker of aPKC activity, and expression of a kinase-defective PKCζ mutant in commissural neurons resulted in A–P randomization in “open-book” explants. Upstream of PKCζ, heterotrimeric G-proteins and phosphatidylinositol-3-kinases (PI3Ks), are also required for A–P guidance, because pertussis toxin, wortmannin, and expression of a p110γ kinase-defective construct all resulted in A–P randomization. Overexpression of p110γ, the catalytic subunit of PI3Kγ, caused precocious anterior turning of commissural axons before midline crossing in open-book explants and caused dissociated precrossing commissural axons, which are normally insensitive to Wnt attraction, to turn toward Wnt4-expressing cells. Therefore, we propose that atypical PKC signaling is required for Wnt-mediated A–P axon guidance and that PI3K can act as a switch to activate Wnt responsiveness during midline crossing.