The　aim　of　this　study　was　to　determine　whether　thymoquinone,　the　principal　active　ingredient　in　the　volatile　oil　of　Nigella　sativa　seeds,　could　suppress　certain　virulence　traits　of　Cronobacter　sakazakii　ATCC　29544　which　contribute　to　infection.　Sub-inhibitory　concentrations　of　thymoquinone　significantly　decreased　motility,　quorum　sensing,　and　endotoxin　production　of　C.　sakazakii　ATCC　29544　and　biofilm　formation　of　C.　sakazakii　7-17.　Thymoquinone　substantially　reduced　the　adhesion　and　invasion　of　C.　sakazakii　ATCC　29544　to　HT-29　cells　and　decreased　the　number　of　intracellular　bacterial　cells　within　the　RAW　264.7　macrophage　cells.　Thymoquinone　also　repressed　the　transcription　of　sixteen　genes　involved　in　the　virulence.　These　findings　suggest　that　thymoquinone　could　attenuated　virulence-related　traits　of　C.　sakazakii　ATCC　29544,　and　its　effects　on　other　C.　sakazakii　strains　and　in　vivo　C.　sakazakii　infection　need　further　investigation.