Cronobacter sakazakii is a gram-negative opportunistic pathogen, which can cause local necrotizing enterocolitis, bacteremia and meningitis. Powdered infant formula is considered to be the major transmission route for newborn infections. The ability of C. sakazakii to grow at refrigerated temperatures appears to be critical in the transmission. In order to understand its molecular mechanism for low temperature growth, we constructed transposon random mutant library and screened for decreased growth at 8oC. Plasmid recovery technique and bioinformatics analysis were used to locate and identify the insertion sites in mutants. Furthermore, extrachromosomal genetic complementation of the identified genes completely (or partially) recovered bacterial growth at low temperature. Overall, we report 14 genes that play an important role in the growth of C. sakazakii ATCC 29544 at low temperature.