Pangolins (scaly anteaters, Pholidota) are among those mammals that are most affected by the international, illegal wildlife trade. Recently, wildlife rescue centers in China became dedicated to rehabilitate confiscated pangolins and prepare them for reintroduction to the wild. Chronic stress is thought to be the main reason for a disturbed microbiota community and a higher mortality rate of pangolin in captivity. In this study, we compared the cortisol levels and the fecal microbiome of Malayan pangolin (Manis javanica) born and reared in captivity (PCB; n = 7) with those rescued from the wildlife trade (PCT; n = 16). Results show that the level of cortisol in PCT was significantly lower than that observed in PCB. There were also significant differences in the composition of the fecal microflora between the two groups, and the diversity of intestinal microbiota was higher in PCB than in PCT. At the phylum level, the bacteria with significant difference between the two groups included Firmicutes and Bacteroides. At the genus level, bacteria such as Bacteroides, Parabacterides, and Clostridium showed significant differences between the two groups. This study proves that chronic stress has a considerable effect on the diversity and composition of fecal microbiota in Malayan pangolin.