This study hypothesized that dietary feed additive containing probiotics alter either immune-related serum substances or serum metabolites in Hanwoo heifers. A probiotic treatment was given at 0.5% top-dressing of concentrate diet for 6 months. The change of immunological indicators in the blood was analyzed under LPS (Lipopolysaccharide) challenge. One day before administration of LPS, all heifers were fitted with an indwelling jugular vein catheter for serial blood collections. Both a serum tube and an EDTA-coated tube were collected at 30-min intervals from-2 to 8 hours relative to the LPS challenge at time 0 (1 ㎍/㎏ of BW). Serum was used for analyzing albumin (ALB), glucose (GLU), total protein (TP), triglycerides (TG), phosphorus (IP), and non-esterified fatty acids (NEFA). Plasma was used for analyzing white blood cell (WBC), red blood cell (RBC), platelet (PLT) and inflammation-related factors (NE, LY, MO, EO, BA, Hb, HCT, MCV, MCH, MCHC, RDW, MPV). There were significant differences in ALB, GLU, TG, IP, and NEFA concentration with the passage of hours post challenge (p < 0.05). The level of ALB, GLU, TG, and IP showed significant difference (p < 0.05) between treatments. However, none of the data showed interaction between time and treatments (p > 0.05). The level of WBC, EO, LY, and MO were reduced after LPS challenge (p > 0.05). In conclusion, LPS challenge after dietary supplementation of probiotics changed the levels of both serum metabolites and inflammationrelated factors. The increase of GLU and TG indicated a probiotics-positive response under LPS challenge (p < 0.05).