It is generally accepted that live attenuated influenza vaccine (LAIV) has the potential for use as a vaccination against flu. In this study, we demonstrated the nature of an influenza A virus (IAV) mutant induced by treating the IAV with a stable furan derivative, (1R,2R)-1-(5'-methylfur-3'-yl)propane-1,2,3-triol (MFPT), which had been isolated from Streptomyces sp. strain FV60 with the objective of it being an LAIV candidate. The resulting MFPT-resistant (MFPTr) IAVs possessed attenuated pathogenicity in vitro and in vivo when compared with that of the parent virus (H1N1 subtype, NWS strain). Sequencing analysis revealed that a novel mutation, C490U in ns gene (P164S in NS1), was detected in all MFPTr virus clones tested. Therefore, NS1 might be a main target of MFPT, and it was suggested that the P164S mutation contributed to the attenuated pathogenicity of the mutants. Although the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is one of the targets of NS1, the MFPTr virus suppressed the phosphorylation of Akt when compared with the wild-type (WT) virus. It was suggested that this might lead to the subsequent inhibition of the cleavage of PARP-1 and caspase-3, which is important for the progression of apoptosis. At the same time, nucleoprotein (NP) was found to be retained in the nuclei in MFPTr virus-infected cells while nuclear export of NP was detected in WT virus-infected cells. In addition, the expression levels of interferon-β transcripts were significantly decreased in MFPTr virus-infected cells. From these results it can be shown that the mutation, NS1P164S, might be one of the key residues to control NS1 function concerning the induction of apoptosis. In conclusion, MFPT induced favorable mutation in the ns gene for the attenuation of IAV, and therefore might provide the novel methodology for preparing LAIVs.