Nicotinic stimulation of the α7 acetylcholine receptors (α7AChRs) mitigates the lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) and other cytokines release in macrophages. This effect is blocked by α7AChR antagonist, α-bungarotoxin (BTX). We tested and confirmed the hypotheses that LPS upregulates α7AChRs, and the prototypical α7AChR antagonists, vecuronium and BTX, do not block the effects of GTS-21, a specific α7AChR agonist, on TNF-α release. With the knockdown of α7AChR expression by short interference RNA, GTS-21 effects on inhibition of TNF-α release were not demonstrable. In addition, GTS-21 mitigated the LPS-induced growth arrest of macrophages in vitro in J774A.1 cells and ex vivo in peritoneal macrophages obtained from mice at 3 days after burn. Moreover, GTS-21 reduced mortality after burn injury in mice. These results indicate that (i) LPS upregulates α7AChRs; (ii) the therapeutic beneficial effects of GTS-21 on cytokine release are specifically mediated via α7AChRs and are preserved even when cotreated with prototypical antagonist, BTX, or clinically used muscle nicotinic antagonist, vecuronium; (iii) activation of α7AChRs by GTS-21 partially reverses the LPS-induced proliferation arrest; and (iv) GTS-21 reduces mortality in mice with burn injury. The in vivo beneficial effects of GTS-21 in burn injury warrant further studies.