Fertilized hen egg is traditionally considered as a dietary supplement in many Asian countries. This work aimed to obtain information on the effect of fertilization on total nutritional composition of egg contents and protein conformation. Chemical analysis showed that the lipid level in fertilized egg began to decrease after day 9. Fertilized egg before day 9 was higher in essential free amino acids (EFAA) and monounsaturated fatty acids (MUFA) contents but lower in cholesterol and polyunsaturated fatty acids (PUFA) than unfertilized counterparts. Fertilized egg proteins were characterized by an increase in hydrophobicity and a decrease in electrostatic interaction. Circular dichroism analysis showed that β-sheet decreased with increasing incubation time, whereas unordered structure increased. The findings observed in this work provide a crucial basis for understanding nutritional composition and protein conformation of fertilized egg, with the potential of being utilized as an EFAA/MUFA-rich, low-cholesterol dietary supplement.