Obesity has become a worldwide epidemic that causes both health and economic burdens. The mild but persistent elevation of the bacterial product lipopolysaccharide (LPS) in the systemic circulation contributes to increased risk and progression of many obesity-associated chronic pathologies. Galectin-3 (Gal3), a mediator of inflammation, is up-regulated in obesity and regulates LPS-induced inflammation, while LPS also influences Gal3 production both in vivo and in vitro. Therefore, we designed this study to investigate the association between Gal3 with markers of adiposity and inflammation in metabolically healthy women who are predominantly obese and African-American, as well as the reciprocal regulation of Gal3 and LPS in mice. We found that serum Gal3 levels were significantly higher in obese women compared to the non-obese individuals but comparable among different classes of obesity. Markers of adiposity and inflammation significantly and positively influenced Gal3 levels only in women with low/moderate risk of cardiovascular disease (CVD). In mice, Gal3 deficiency led to induction of significantly lower circulating cytokines and hepatic acute-phase response caused by non-lethal doses of LPS administration. Additionally, inhibition of extracellular Gal3 did not replicate the protective effects of Gal3 mentioned above, which were specific for LPS (TLR4)-induced inflammation, as we observed opposite outcomes in Poly I:C (TLR3) injected mice. Moreover, LPS differentially affected protein production versus gene expression of Gal3 both in vivo and in vitro. Finally, repeated injections of ultra-low doses of LPS, which mimics the mild endotoxemia in obesity, induced a trend towards increased adiposity and development of mild hyperglycemia, as well as increased hepatic acute-phase response in wild-type mice. While those LPS-induced alterations were either less remarkable, or absent in Gal3 knockout mice. Together, our findings indicate that Gal3 positively associates with adiposity and inflammation in women, while presence of elevated CVD risk may weaken the strength of Gal3 as a biomarker of inflammation. Additionally, Gal3 specifically promotes LPS-induced inflammation in mice, as Gal3 deficiency protects mice from both non-lethal and repeated ultra-low doses of LPS-induced inflammation, while LPS also affects production of Gal3 in a complex manner.