Objective A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons.However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.Methods In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated.Results (1) Forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes 1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth; (2) Inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis; (3) These Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj 1 and specific commissural markers TAG1 and Pax7 was unchanged.Conclusion We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.