The effect of a DNA immunostimulant on inflammatory and immune responses, performance, and health in calves following abrupt weaning and introduction to a concentrate diet was tested. Sixty-four single source Angus crossbred steers were weaned on day 1 and assigned to receive a DNA immunostimulant (TRT) or saline (CON) on days 0, 2, 4, and 6. On day 0, steers received clostridial and respiratory vaccines and anthelmintic; they were then transported 2 h, allocated to pens (n = 8 per pen), and introduced to total mixed ration. Daily intake, ADG, and feed efficiency were measured. Serum haptoglobin, tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) were assayed by ELISA or AlphaLISA on days 0, 2, 4, 6, 14, and 28; serum-neutralizing antibodies (SNA) to bovine herpesvirus-1 and bovine viral diarrhea virus-1 (BVDV-1) were quantified on days 0, 28, 68, and 135. In a subset of cattle (n = 6 to 8 per treatment group), the percent macrophages and activated gamma delta (γδ) T cells in blood was determined by flow cytometry on days 2 and 6, and expression of mRNA for TNF-α, interferon-gamma (IFN-γ), IL-4, and IL-10 by stimulated blood mononuclear cells was assessed by real-time reverse transcriptase PCR on day 6. After 70 d, cattle were shipped 1,205 km to a feedlot and performance and health were followed. There was a significant effect of time on serum TNF-α, IL-1β, haptoglobin, and SNA (P < 0.001); the range in concentration among cattle on each day was large. The ratio of IFN-γ to IL-4 expression was significantly higher (P = 0.03) for TRT cattle, suggesting that treatment activated T-helper type 1 cells. There was a trend toward an improved feed conversion (P = 0.10) for TRT steers over the 70-d backgrounding period. There was no effect of treatment on feedlot performance or carcass merit (P > 0.10). During backgrounding, 1 TRT steer died of enterocolitis. In spite of backgrounding, cattle experienced an outbreak of bovine respiratory disease (BRD) in the feedlot and 1 of 31 TRT cattle and 5 of 32 CON cattle died of BRD. The immunostimulant modified some immune responses during backgrounding. Large variability in inflammatory responses during backgrounding indicated that events around weaning induce systemic inflammation that varies substantially among cattle.