Abstract: 【Objective】 This study systematically investigated the effects of replacing fish meal（FM） with Chlorella powder on Litopenaeus vannamei and explored the underlying mechanisms, which provided theoretical basis for the application of Chlorella in formulated feed for Litopenaeus vannamei. 【Method】 Litopenaeus vannamei was fed by four isonitrogenous and isolipidic feeds which were prepared by replacing 0%, 15%, 30% and 45% of FM in the basal 3 with Chlorella powder, designated as CHL15 group, CHL30 group and CHL45 group respectively, with basic feed as control（FM group）. Each group had 3 replicates, with 40 Litopenaeus vannamei per replicate. After 8 week of cultivation, the effects of Chlorella powder replacing FM on Litopenaeus vannamei were evaluated across multiple dimensions, including the growth performance, disease resistance, immune reaction, digestive function, transcriptome level, and intestinal microbiota composition. 【Result】 When the replacement rate of FM with Chlorella powder reached 45%, the Litopenaeus vannamei showed significantly decreased weight gain rate（WGR）, specific growth rate（SGR） and protein efficiency ratio（PER）（P＜0.05, the same below）, along with significantly increased feed conversion ratio（FCR） compared to the control group. After Vibrio parahaemolyticus challenge, the survival rates of Litopenaeus vannamei in all the CHL groups were significantly higher than that in FM group. Compared with the FM group, all the CHL groups demonstrated significantly increased activities of phenoloxidase（PO）, superoxide dismutase（SOD） and lysozyme（LZM） in hepatopancreas, as well as significantly higher trypsin and amylase activities in intestine. There were 112, 852 and 748 differentially expressed genes（DEGs） in CHL15, CHL30 and CHL45 groups respectively compared to FM group, with 20 shared DEGs identified. The DEGs were primarily enriched in signaling pathways related to metabolism. The replacement of FM by Chlorella powder significantly reduced the ACE index and Chao1 index of intestinal microbiota, while the Shannon index and Simpson index remained unchanged significantly（P＞0.05）. The relative abundances of Vibrio and Tenacibaculum significantly lower than those in the FM group, whereas the relative abundances of Ruegeria was significantly higher than FM group. Tax4Fun functional prediction suggested that Chlorella might enhance intestinal homeostasis in Litopenaeus vannamei by regulating the synthesis of secondary metabolites（terpenoids） and environmental adaptation mechanisms. Pearson correlation analysis revealed that Chlorella modulated nutrient absorption and metabolism in Litopenaeus vannamei by influencing interactions between intestinal microbiota and key metabolic genes. 【Conclusion】 Chlorella enhances the immunity and digestive capacity of Litopenaeus vannamei by regulating metabolic pathways and optimizing intestinal microbiota structure. Replacing suitable FM（15%-30%） with Chlorella powder in feed is feasible, but excessive substitution should be avoided.