In this study, Litopenaeus vannamei was fed by the feed using Clostridium autoethanogenum protein (CAP) as a single protein source to replace fish meal (FM) at different salinity, aiming to explore the effects of salinity stress on shrimp fed with new protein source. L. vannamei were fed one of two isoproteic and isolipidic diets of differing protein sources (FM or CAP) for 8 weeks while being maintained at three different salinity (15 ppt, 30 ppt or 45 ppt). The intestinal microbiota analysis showed that the alpha diversity of L. vannamei was significantly affected by salinity or protein sources. Generally speaking, CAP could increase the relative abundance of beneficial bacteria while decrease the relative abundance of harmful bacteria in the intestine of L. vannamei at phylum, family and genus levels, and it was more affected by salinity changes when compared with FM. Besides, the changes of salinity and protein sources lead to different changes in intestinal microflora function of L. vannamei. In sum, this study indicated that CAP could improve intestinal microflora diversity of L. vannamei, and enhance its ability to cope with salinity stress.