The effects of mother-infant separation (i.e., weaning) on the physiology, psychology and nutrition of mammalian infants have attracted much attention. Forest musk deer (FMD) is a first-class protected species in China and listed endangered in the IUCN Red List. The captive breeding population is not only an important source for restocking of wild resources, but also a necessary way to supply the market with legal musk. So far, there is no scientific basis for the appropriate separation time of FMD females and their infants. Therefore, we used metagenome sequencing and enzyme-linked immunosorbent assays to study changes in the fecal cortisol concentration, as well as the intestinal microbiome composition and function of females and fawns at three different separation times, i.e., after 80 days, 90 days and 100 days. The results showed that the increment of the cortisol concentration in female FMD increased with increasing lactation time. The increment of cortisol concentration in infant FMD was highest in the 80 days weaning group, but there was no significant difference between the 90 days and the 100 days separation time. Based on the annotation results of COG, KEGG and CAZy databases, the abundance of different functions annotated by the intestinal microbiome of mothers and fawns of the 90 days weaning group changed slightly after separation. Based on the above results, the separation of mother and infant FMD is recommended after 90 days, i.e., the separation time that triggered the lowest rate of weaning stress and that supported a relatively stable gastro-intestinal physiology.