An intramammary challenge with lipopolysaccharide (LPS) from Escherichia coli is known to induce a considerable immune response of the mammary gland and an impairment of the blood-milk barrier. The aim of the current study was to investigate a potential immune reaction in the mammary gland and potential changes of the integrity of the blood-milk barrier in response to an intravenous, i.e., systemic challenge, with LPS. Ten lactating dairy cows in wk 3 of lactation were challenged intravenously (V. jugularis) with 0.5 µg/kg BW E. coli LPS (O26:B6). Rectal temperature was measured hourly. Milk samples of one udder quarter were taken immediately before and then every 30 min until 5 h after challenge. Mammary gland biopsies of one quarter were taken immediately before and 8 h after LPS challenge for RT-qPCR of immunorelevant factors. Rectal temperature increased (P < 0.001) within 1 h of LPS administration from 38.3 ± 0.1 to 39.2 ± 0.1°C and stayed elevated throughout the 5 h of experiment. The milk somatic cell count was 83.3 ± 19.1 × 10³/mL immediately before challenge and did not change throughout the experiment. Lactate-dehydrogenase concentrations in milk as a marker for blood-milk barrier impairment was 48.1 ± 6.0 U/L immediately before challenge and did not change throughout the experiment. Relative mRNA expression of immunorelevant factors; that is, the cytokines TNF-α and interleukin-1β in mammary gland tissue did not change in response to intravenous LPS injection. In conclusion, in contrast to intramammary injections, an intravenous injection of 0.5 µg/kg BW LPS induces a systemic immune response shown by an increase in rectal temperature, but has obviously no effects on factors of mammary gland immune response which typically change during LPS-induced mastitis. Also the blood-milk barrier integrity does not appear to be influenced by systemic LPS.