Influenza neuraminidase (NA) is a pivotal target for viral infection control. However, the accumulating of mutations compromise the efficacy of NA inhibitors. Thus, it is critical to design new drugs targeted to different motifs of NA. Recently, a new motif called 340-cavity was discovered in NA subtypes close to the calcium binding site. The presence of calcium is known to influence NA activity and thermostability. Therefore, the 340-cavity is a putative ligand-binding site for affecting the normal function of NA. In this study, we performed molecular dynamics simulations of different NA subtypes to explore the mechanism of 340-loop formation. Ligand-binding site prediction and fragment library screening were also carried out to provide evidence for the 340-cavity as a druggable pocket. We found that residues G342 and P/R344 in the 340-loop determine the size of the 340-cavity, and the calcium ion plays an important role in maintaining the conformation of the 340-loop through contacts with G345 and Q347. In addition, the 340-cavity is predicted to be a ligand-binding site by metaPocket, and a sequence analysis method is proposed to predict the existence of the 340-cavity. Our study shows that the 340-cavity is not an occasional or atypical domain in NA subtypes, and it has potential to function as a new hotspot for influenza drug binding. (C) 2016 Elsevier Inc. All rights reserved.