Highly pathogenic avian influenza A H5N1 virus causes pneumonia and acute respiratory distress syndrome in humans. Virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. Galectin-3, a β-galactoside-binding protein widely distributed in immune and epithelial cells, regulates various immune functions and modulates microbial infections. Here we describe galectin-3 up-regulation in mouse lung tissue following challenges with the H5N1 influenza virus. We investigated the effects of endogenous galectin-3 on H5N1 infection and found that survival of galectin-3 knockout (Gal-3KO) mice was comparable to wild-type (WT) mice following infections. Compared to infected WT mice, infected Gal-3KO mice exhibited less inflammation in the lungs and reduced interleukin-1 beta (IL-1β) levels in bronchoalveolar lavage fluid. Also, the bone marrow–derived macrophages (BMMs) from Gal-3KO mice exhibited reduced oligomerization of apoptosis-associated speck-like proteins containing caspase-associated recruitment domains, and secreted less IL-1β compared to BMMs from WT mice. However, similar levels of the inflammasome component of nucleotide oligomerization domain–like receptor protein 3 (NLRP3) were observed in two genotypes of BMMs. Co-immunoprecipitation data indicated galectin-3 and NLRP3 interaction in BMMs infected with H5N1. An association was also observed between galectin-3 and NLRP3-ASC complex. Combined, our results suggest that endogenous galectin-3 enhances the effects of H5N1 infection by promoting host inflammatory responses and regulating IL-1β production by macrophages via interaction with NLRP3.