Enterobacter sakazakii is considered an opportunistic foodborne pathogen that is characterized by formation of yellow-pigmented colonies. Because of the lack of basic knowledge about Enterobacter sakazakii genetics, the BAC approach and the heterologous expression of the pigment in Escherichia coli were used to elucidate the molecular structure of the genes responsible for pigment production in Enterobacter sakazakii strain ES5. Sequencing and annotation of a 33.025 bp fragment revealed seven ORFs that could be assigned to the carotenoid biosynthesis pathway. The gene cluster had the organization crtE-idi-XYIBZ, with the crtE-idi-XYIB genes putatively transcribed as an operon and the crtZ gene transcribed in the opposite orientation. The carotenogenic nature of the pigment of Enterobacter sakazakii wt was ascertained by in situ analysis using visible microspectroscopy and resonance Raman microspectroscopy.