The A(H1N1)pdm09 virus, which caused the 2009 influenza pandemic, has continued to circulate in humans for over a decade. Understanding its biological properties is crucial for effective surveillance, prevention, and control. Here, we characterized recently circulating A(H1N1)pdm09 viruses, focusing on strains isolated between 2016 and 2019. HA gene-based phylogenetic tree analysis revealed that post-pandemic A(H1N1)pdm09 virus strains circulating between 2016 and 2019 form two clusters: subclade 6B.1 and subclade 6B.1 A.5a. Growth kinetics of nine selected representative strains from these clusters showed that subclade 6B.1 viruses replicated well in human lung cells, whereas some subclade 6B.1 A.5a viruses replicated poorly. In vivo, all viruses from both subclades caused significantly less weight loss in infected mice compared to the prototypic pandemic strain A/California/04/2009 (Cal04/2009). Additionally, virus titers in the lungs of mice infected with most viruses from subclade 6B.1 or 6B.1 A.5a were significantly lower than those in mice infected with Cal04/2009. Furthermore, evolutionary analysis suggested multiple transitions to a less pathogenic phenotype, indicating an evolutionary trend towards attenuation. These results demonstrate that A(H1N1)pdm09 viruses isolated between 2016 and 2019 are attenuated in mice, although the mutations responsible for this attenuation require further investigation. Our findings emphasize the need for continued monitoring of A(H1N1)pdm09 viruses to understand their evolutionary dynamics and potential impact on public health.