Gut microbiota is one of the major elements in the control of host health. However, the composition of gut microbiota in koalas has rarely been investigated. Here, we performed 16S rRNA gene sequencing to determine the individual and environmental determinants of gut microbiota diversity and function in 35 fecal samples collected from captive koalas. Meanwhile, blood immune-related cytokine levels were examined by quantitative reverse transcription-PCR to initially explore the relationship between the gut microbiota and the immune system in koalas. The relative abundance of many bacteria, such as Lonepinella koalarum, varies at different ages in koalas and decreases with age. Conversely, Ruminococcus flavefaciens increases with age. Moreover, bacterial pathways involved in lipid metabolism, the biosynthesis of other secondary metabolites, and infectious disease show a significant correlation with age. Age affects the relationship between the microbiota and the host immune system. Among them, the gut microbiota of subadult and aged koalas was closely correlated with CD8β and CD4, whereas adult koalas were correlated with CLEC4E. We also found that sex, reproductive status, and living environment have little impact on the koala gut microbiota and immune system. These results shed suggest age is a key factor affecting gut microbiota and immunity in captive koalas and thus provide new insight into its role in host development and the host immune system. IMPORTANCE Although we have a preliminary understanding of the gut microbiota of koalas, we lack insight into which factors potentially impact captive koalas. This study creates the largest koala gut microbiota data set in China to date and describes several factors that may affect gut microbiota and the immune system in captive koalas, highlighting that age may be a key factor affecting captive koalas. Moreover, this study is the first to characterize the correlation between gut microbiota and cytokines in koalas. Better treatment strategies for infectious disorders may be possible if we can better understand the interactions between the immune system and the microbiota.