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The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors, and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement (referring both attachment and metamorphosis in this paper hereafter) in a dose-dependent manner. In contrast, both NO scavenger 2-(-4-Carboxypheny)-4,4,5,5-tetramethylimidazolineoxyl-1-oxyl-3-oxide (Carboxy-PTIO) and NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream GC activity by using a GC-selective inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) could also significantly accelerate the larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a critical stage for the larval decision of attachment and metamorphosis. In sum, we concluded that NO regulates larval settlement via mediating down-stream cGMP signalling.