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The popularity of sea cucumbers has led to overexploitation of wild populations and increasing need to develop their aquaculture. In vitro induction of oocyte maturation followed by fertilization represents an innovative approach that could help increase their production. The inducers of oocyte maturation are naturally synthesised within neural tissue, which in sea cucumbers includes longitudinal radial nerves and a circumoral nerve ring. In this study, we showed that reproductive maturation of the black sea cucumber (Holothuria leucospilota Brandt, 1835) in South-East Queensland, Australia, peaked in December, based on biometric analysis of gonads. Radial nerve extract (RNE; 233 μg/ml) prepared from reproductive mature females induced in vitro oocyte maturation at 98.9% efficiency, based on germinal vesicle breakdown. RNE-matured oocytes were fertilized (99%) with conspecific sperm and larvae developed to late auricularia stage (25 days post-fertilization). The NGIWYamide neuropeptide (cubifrin), which induces oocyte maturation in the sea cucumber Apostichopus japonicus, could not induce oocyte maturation in H. leucospilota. These results provide information that could be used for large-scale artificial breeding of H. leucospilota, and a platform towards the identification of the bioactive RNE factor(s).