Laminitis is one of the most common diseases in horses and affects the equine hoof. The pathogenesis of laminitis is yet not fully understood. Several different trigger factors for laminitis are discussed. Among them, endotoxins, also called lipopolysaccharides (LPS), seem to play an important role during the development of laminitis. Therefore, an ex vivo/in vitro explant hoof model was used to test the effect of LPS [0 - 200 g/mL] for 24 and 48 hours on explant integrity. Glucose, acetic -, lactic -, and propionic acid concentrations in explant supernatants were measured to evaluate energy metabolism of the hoof tissue. Furthermore, dermal and epidermal cells were isolated from the equine hoof to test cytotoxicity of LPS. In addition, it was tested if Polymyxin B (PMB), a milk thistle extract (MT), or Silymarin can directly deactivate LPS or if they are able to inhibit LPS-induced lamellar separation in equine hoof explants. LPS significantly increased the numbers of separated explants after 24 [5 - 200 g/mL] and 48 [20 - 200 g/mL] hours. The force needed to separate explants was significantly decreased when explants were incubated with 10 and 100 g/mL LPS. LPS led to significantly decreased lactic acid concentrations in explants incubated with 5, 10, or 100 g/mL LPS. There was no effect of LPS on the viability of epidermal and dermal hoof cells. LPS was directly neutralized by Silymarin (75%) in a similar way than PMB (97%). PMB, Silymarin, and MT significantly improved separation pattern and separation force of explants incubated with 10 g/mL LPS. As endotoxins induce lamellar separation of hoof explants in vitro, the influence of endotoxins should be further investigated for their contribution during the pathogenesis of laminitis. Silymarin and MT were able to reduce endotoxin activity in vitro and eliminate the effects of LPS on the hoof tissue. Thus, silymarin or MT might be used to prevent laminitis and reduce endotoxin induced effects.