ABSTRACTGiven the growing evidence that gut dysfunction, including changes in gut microbiota composition, plays a critical role in the development of inflammation and metabolic diseases, the identification of novel probiotic bacteria with immunometabolic properties has recently attracted more attention. Herein, bacterial strains were first isolated from dairy products and human feces and then screened in vitro for their immunomodulatory activity. Five selected strains were further analyzed in vivo, using a mouse model of diet‐induced obesity. C57BL/6 mice were fed a high‐fat high‐sucrose diet, in combination with 1 of 3 Lactobacillus strains (Lb38, L. plantarum; L79, L. paracaseil casei; Lbl02, L. rhamnosus) or Bifidobacterium strains (Bf26, Bfl41, 2 different strains of B. animalis ssp. lactis species) administered for 8 wk at 109 colony‐forming units/d. Whereas 3 strains showed only modest (Lb38, Bf26) or no (L79) effects, Lbl02 and Bfl41 reduced diet‐induced obesity, visceral fat accretion, and inflammation, concomitant with improvement of glucose tolerance and insulin sensitivity. Further analysis revealed that Lbl02 and Bf141 enhanced intestinal integrity markers in association with selective changes in gut microbiota composition. We have thus identified 2 new potential probiotic bacterial strains with immunometabolic properties to alleviate obesity development and associated metabolic disturbances.—Le Barz, M., Daniel, N., Varin, T. V., Naimi, S., Demers‐Mathieu, V., Pilon, G., Audy, J., Laurin, E., Roy, D., Urdaci, M. C., St‐Gelais, D., Fliss, I, Marette, A. In vivo screening of multiple bacterial strains identifies Lactobacillus rhamnosus Lbl02 and Bifidobacterium animalis ssp. lactis Bf 141 as probiotics that improve metabolic disorders in a mouse model of obesity. FASEB J. 33, 4921—4935 (2019). www.fasebj.org