Host Defense Peptides (HDPs) such as cathelicidins are multifunctional effectors of the innate immune system with both antimicrobial and pleiotropic immunomodulatory functions. Chicken cathelicidin-2 (CATH-2) has multiple immunomodulatory effects in vitro and the D-amino acid analog of this peptide has been shown to partially protect young chicks from a bacterial infection. However, the mechanisms responsible for CATH-2 mediated in vivo protection have not been investigated so far. In this study, D-CATH-2 was administered in ovo and the immune status and microbiota of the chicks were investigated at 7 days posthatch to elucidate the in vivo mechanisms of the peptide. In three consecutive studies, no effects on numbers and functions of immune cells were found and only small changes were seen in gene expression of Peripheral Blood Mononuclear Cells (PBMCs). In two studies, intestinal microbiota composition was determined which was highly variable, suggesting that it was strongly influenced by environmental factors. In both studies, in ovo D-CATH-2 treatment caused significant reduction of Ruminococcaceae and Butyricicoccus in the cecum and Escherichia/Shigella in both ileum and cecum. In conclusion, this study shows that, in the absence of an infectious stimulus, in ovo administration of a CATH-2 analog alters the microbiota composition but does not affect the chicks' immune system posthatch.