Vibrio natriegens has massive biotechnological potential owing to its fast growth rate. However, this bacterium rapidly loses its culturability during low-temperature preservation (LTP), the reason for which is still unknown. To reveal the metabolic responses of V. natriegens during LTP, we analyzed and compared the transcriptome before and after 8 days of preservation at 4 or 25 °C (room-temperature preservation (RTP)) in liquid culture medium. Most genes exhibited significant transcriptional responses to LTP. Using gene set enrichment analysis, we compared the transcriptional responses of different V. natriegens Gene Ontology (GO) sets during LTP or RTP. The enrichment of the GO set "SOS response" during LTP, but not RTP, indicated the occurrence of DNA damage during LTP. The GO set "respiratory electron transport chain" was suppressed during LTP and RTP. Although the GO set "response to oxidative stress" was not significantly altered, we observed an increase in reactive oxygen species (ROS) during LTP, suggesting a relationship between ROS and cold-induced loss of culturability (CILC) in V. natriegens. The faster loss of culturability and accumulation of ROS in 20 mL compared to 100 mL of liquid culture medium further suggested a relationship between CILC and oxygen availability. Furthermore, we showed that the deletion of Na+-translocating NADH-ubiquinone oxidoreductase, but not type-II NADH dehydrogenase, accelerated CILC and increased intracellular ROS levels in V. natriegens. These findings will help to understand the cause of CILC which may lead to improving the stability of V. natriegens at low temperatures.