Activation of c-Jun terminal kinase (JNK) by the nonstructural protein 1 (NS1) of the H5N1 subtype of influenza A virus (IAV) plays an important role in inducing autophagy and virus replication. However, the mechanisms of NS1-induced JNK activation remain elusive. Here we first confirmed the ability of H5N1 (A/mallard/Huadong/S/2005) to activate JNK and to induce autophagy in 293T cells, a human embryonic kidney cell line. We further showed that TAK1, MAP kinase kinase 4 (MKK4), and JNK were activated in 293T cells transfected with the NS1 gene of the H5N1 virus. JNK activation by the NS1 protein or by H5N1 virus was blocked by 5Z-7-Oxozeaenol (5Z), a TAK1-specific inhibitor, and by TAK1 siRNA. Further study showed that 5Z and TAK1 siRNA suppressed H5N1 virus-induced autophagy and inhibited virus replication. Our study unveiled a previously unrecognized role of TAK1 in IAV replication, IAV-induced JNK activation, and autophagy.