Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the -independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation.