The OmpW family consists of a ubiquitous group of small outer membrane (OM) β-barrel proteins of Gram-negative bacteria with proposed roles in environmental adaptation but poorly understood mechanisms of expression. We report here that Escherichia coli K-12 OmpW contents are drastically modified by temperature changes compatible with the leap from the environment to warm-blooded hosts and/or vice versa. Thus, while OmpW is present in the OM of bacteria grown at 37 °C, it sharply disappears at 23 °C with the concomitant acquisition of colicin S4 resistance by the cells. ompW::lacZY fusions indicated that temperature regulation operates at the level of transcription, being ompW expression almost abolished at 23 °C as compared to 37 °C. Moreover, E. coli Δhns mutants lacking H-NS showed reductions in ompW transcription and OmpW contents at 37 °C, indicating positive modulatory roles for this nucleoid-structuring protein in ompW expression. Also, ΔhnsΔstpA double mutants simultaneously lacking H-NS and its paralog StpA showed more severe reductions in ompW expression at 37 °C, resulting in the complete loss of OmpW. The overall results indicate that OmpW contents in E. coli are regulated by both temperature and H-NS and reinforce OmpW functions in bacterial adaptation to warm-blooded hosts.