In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.