Bifidobacterium animalis ssp. lactis Probio-M8 is a potential probiotic strain that was isolated from human milk. Previously, we obtained an oxygen-resistant variant (Probio-M8o) of Probio-M8 by an adaptive evolution strategy. In the present study, a comparative transcriptomic analysis of Probio-M8o and Probio-M8 was carried out to reveal the cellular mechanism of the oxygen-resistant phenotype. Using RNA-seq, 210 and 217 differentially expressed genes were identified in Probio-M8o compared with Probio-M8 after oxygen exposure for 30 and 60 min, respectively. The oxygen treatment upregulated a set of genes that encoded proteins responsible for fatty acid biosynthesis. This observation was in good agreement with the composition change in fatty acids at the biochemical level. Our study showed that the oxygen-resistant phenotype could be related to adaptation of fatty acid metabolism.