The microbiological safety of avian eggs is a major concern for the poultry industry and for consumers due to the potential for severe impacts on public health. Innate immune defense is formed by proteins with antimicrobial and immune-modulatory activities and ensures the protection of the chick embryo against pathogens. The objective of this project was to identify the chicken eggshell membrane (ESM) proteins that play a role in these innate immune defense mechanisms. We hypothesized that ESM Ovocalyxin-36 (OCX-36) is a pattern recognition protein, and characterized purified ESM OCX-36. OCX-36 has antimicrobial activity against S. aureus and binds E. coli lipopolysaccharide (LPS) and S. aureus lipoteichoic acid (LTA). We additionally investigated the OCX-36 nonsynonymous single nucleotide polymorphisms (SNPs) at cDNA position 211. The corresponding isoforms (proline-71 or serine-71) were purified from eggs collected from genotyped homozygous hens. A significant difference between Pro-71 and Ser-71 OCX-36s for S. aureus LTA binding activity was observed. From these experiments, we confirmed the hypothesis that OCX-36 is a pattern recognition molecule. We also found that OCX-36 has anti-endotoxin properties and is a macrophage immunostimulator to produce NO and TNF-α. Digested OCX-36 down-regulated the expression of genes involved in LPS signaling and inflammatory responses. Moreover, OCX-36-derived peptides inhibited the production of LPS-induced pro-inflammatory mediators associated with endotoxemia in vivo. Quantitative proteomics analysis of ESMs was performed to evaluate changes in ESM protein abundance during chick embryonic development. Bioinformatics analysis revealed enrichment of proteins associated with antimicrobial and immune protection, vascularization, calcium mobilization and lipid transport, which are vital for chick embryonic development. In unfertilized eggs, protease inhibitors and antimicrobial proteins were enriched. In summary, the ESMs are enriched in proteins with antimicrobial, antioxidant and immune-modulatory properties, which aid in the development of the chick embryo and protect the embryo and unfertilized egg against pathogen invasion.