Ammonia toxicity can disrupt the intestinal health of aquatic animals. It is important to find substances that alleviate these adverse effects. The present study explored the possible protective role of myo-inositol (MI) in ammonia-induced toxicity in the fish intestine. Great blue-spotted mudskippers (Boleophthalmus pectinirostris) accumulated in artificial seawater (15‰ salinity, n = 600) were randomly selected and intraperitoneally injected with NaCl (0.68%) or MI (2.5 mg/g fish in 0.68% NaCl) then exposed to artificial seawater alone (NaCl and MI group) or seawater containing 57.025 mmol/L ammonium chloride (NH3 and NH3 + MI group). After a 24-h experiment, it showed that ammonia exposure down-regulated the mRNA expression levels of intestinal barrier function proteins (Zo-1, Ocln, Cldn-5, Cldn-12, and Cldn-15) and anti-inflammatory cytokines (Tgf-β and Il-10) while the acute ammonia stress up-regulated the apoptosis genes (p53, Bax, Caspase-3, and Caspase-9) and pro-inflammatory cytokines (Tnf-α and Il-1β). Furthermore, ammonia challenge also induced oxidative stress, as the malondialdehyde and the protein carbonyl contents were increased. In addition, ammonia stress down-regulated the antioxidant enzymes (Cu/Zn-Sod, Cat, Gpx, and Gst) activities as well as their gene transcription levels. The administration of the exogenous myo-inositol greatly ameliorated the ammonia-induced changes in redox capacity, immune response, apoptosis, inflammation, and tight junction barrier function to levels similar to those of the NaCl group. Furthermore, fish injected with MI alone showed no significant changes compared with the NaCl group. Taken together, pretreatment with myo-inositol had no obvious side-effects and effectively protected the mudskippers' intestine from the toxicity caused by acute ammonia stress.