Laminitis is one of the most common causes for lameness in horses and ruminants. The pathology of laminitis is still not fully understood. As it is a multifactorial disease, several substances and toxins such as endotoxins are discussed as possible trigger factors. The aim of our study was to test the influence of endotoxins on the lamellar integrity of hoof explants. Furthermore, the potential difference of effects of endotoxins on the lamellar integrity of hooves from horses and ruminants was investigated. Explants from hooves (n = 3) and claws (n = 3) were cultivated at 37°C and 5% CO2 with D-MEM as culture medium. Lipopolysaccharides (LPS) of Escherichia coli O55:B5 were added to the equine [0, 2.5, 10, 100 µg/mL] and bovine [0, 1, 10, 100 µg/mL] explants for 24 h. After incubation, explants were tested for their integrity by measuring the force [Newton], which is needed to separate the explants ( = separation force). Therefore, the explants were connected to a force transducer with clamps. Viability of explants was tested with the water-soluble tetrazolium (WST-1) assay. There was no effect on separation force when equine explants were incubated with 2.5 µg/mL LPS. Separation force of equine explants incubated with 10 and 100 µg/mL LPS was significantly decreased (P < 0.005) by 45% and 49%, respectively, compared with control explants. Similar to equine explants, there was no effect on separation force when bovine explants were incubated with 1 µg/mL LPS. Separation force of bovine explants incubated with 10 and 100 µg/mL LPS was significantly decreased (P < 0.005) compared with control explants. A reduction by 50% and 65% for LPS was observed, respectively. All explants were viable after 24 h incubation. In our study a concentration dependent reduction of separation force of explants incubated with LPS was observed. Similar effects of LPS were observed in explants from both species. Although laminitis has never been induced by endotoxins alone in animal experiments, our data suggest that endotoxins might play an important role during the onset of laminitis. The presented model could be used to test other potential trigger factors of laminitis and the interaction between these factors.