Adult ewes of 2 breeds (Lacaune, n = 18, 77.4 ± 1.6 kg BW; Manchega, n = 18, 79.3 ± 1.2 kg BW) at late lactation (DIM, 210 ± 5) were used to assess the effects of barley β-glucans (BBG) supplementation on their immune response to a lipopolysaccharide (LPS) intramammary challenge. Ewes were fed alfalfa hay ad libitum and 350 g/d of barley grain cv Hispanic (3.8% BBG; Batlle, Lleida, ES) for 7 d. Thereafter, they were allocated into 3 groups to which the experimental treatments were applied for 14 d. Groups were: 1) Control (CO), fed as during adaptation (13.3 g BBG/d); 2) Oral (OR), fed with a new barley variety (cv Anapurnna, Batlle) of 10% BBG (35 g BBG/d); and, 3) Injected (IN), same as CO and intraperitoneally injected with a single dose of BBG (140 mL of 1.4% BBG; 2 g BBG/ewe). After 9 d, ewes were submitted to an intramammary LPS challenge with 1 mL of an E. coli endotoxin (5 µg/mL, Sigma-Aldrich, St Louis, MI) in an udder-half and 1 mL of saline in the other. Rectal temperature, milk yield, milk composition and plasma interleukin (IL)-1α and IL-1β were monitored daily for 5 d. Data were treated by PROC MIXED for repeated measurements of SAS (v.9.4, SAS Institute Inc., Cary, NC) and LS means separated by PDIFF. No differences by treatment were detected in rectal temperature (P = 0.13), milk yield (P = 0.29) and IL-1α and IL-1β (P = 0.31 to 0.84). However, BBG tended (P = 0.06) to attenuate the lactose decrease in milk by LPS, the decrease being lower in the IN treatment vs. the others (3.80 vs. 3.57%, respectively). The SCC in milk increased due to the effect of LPS, but there were no differences by BBG. The IL-1α and IL-1β did not differ by BBG or time. However, their numerical values increased by time, agreeing with the increase in SCC and inflammation. Moreover, basal IL values were lower (P < 0.01) for IN when compared with the others (299 vs. 352 pg/mL and 79 vs. 152 pg/mL for IL-1α and IL-1β, respectively), indicating that BBG supplementation may increase the immune status and the protection against mastitis. In conclusion, the LPS challenge triggered a short-term immune response in BBG supplemented ewes, which was more effective when injected intraperitoneally. Supported by MINECO/FEDER (AGL2015–69435-C3–3-R).