Poly-β-hydroxybutyrate (PHB) is a microbial storage compound that occurs in the presence of excess carbonsource. PHB is degraded into water-soluble β-hydroxybutyric acid in the gut of aquatic animals and slightly lowers gutpH. The acidic gut environment benefits growth of probiotic bacteria (particularly Gram-positive bacteria), which increasesenzyme secretion and improves nutrient absorption and immunity of the animal. Dietary PHB benefits thegrowth and survival of marine fish and crustaceans, such as European seabass, Dicentrarchus labrax, Siberian sturgeon,Acipenser baerii, and giant freshwater prawn, Macrobrachium rosenbergii. Our previous studies indicated thatPHB-enriched rotifers and Artemia nauplii significantly improve molting, survival, and tolerance to vibriosis challengein Chinese mitten crab, Eriocheir sinesis zoeal larvae, and that PHB supplementation in formulated feed improvesmolting of juvenile E. sinensis at the optimal dietary level of 1% PHB. However, as PHB is an organic acid-releasingcompound, little is known about its possible acidosis effect during long-term feeding or the interaction between feedingduration and dietary PHB level. In this study, the dosing and feeding period effects of dietary PHB on hepatopancreaticbiochemical composition and enzyme activities, as well as intestinal microbial diversity in juvenile E. sinensis, werestudied by feeding formulated diets containing 0%, 3%, 5%, and 10% PHB for 1, 6, 15, and 21 days, respectively. Theresults showed that dietary PHB supplementation affected crude protein content, crude lipid content, soluble proteincontent, and various hepatopancreatic digestive enzymes, as well as microbial diversity in the gastro-intestine, whichwere PHB-dose and feeding-period dependent. After 1 day of feeding, the 10% PHB supplemented group had significantlyhigher total superoxide dismutase (T-SOD) and hepatopancreatic amylase and lipase activities compared withthose in the control group fed a diet containing no PHB (P<0.05). In addition, 5% and 10% PHB resulted in significantlyhigher range-weighted richness (Rr) of the gastro-intestine microbial community (P<0.05). After 6 days of feeding,the 10% PHB treatment resulted in significantly lower amylase activity and the 3% and 5% PHB treatments resultedin significantly lower lipase activity (P<0.05). All PHB supplemented groups had significantly higher Rr values(P<0.05). On day 15, the 10% PHB treatment resulted in significantly lower T-SOD, and the 5% and 10% PHBtreatments resulted in significantly higher lipase activity (P<0.05). All PHB supplemented groups had significantlylower amylase activity but higher Rr values (P<0.05). On day 20, all PHB supplemented groups had significantly reducedT-SOD, amylase, and pepsin activities (P<0.05); the higher the PHB dose, the greater the reduction. The 10%PHB group had a significantly reduced Rr value(P<0.05), whereas 3% and 5% PHB had no effect on the Rrvalue(P>0.05). These results suggest that a longer feeding period could be applied with a lower PHB dose and viceversa to support use of dietary PHB in cultured E. sinensis.