In this work, we demonstrated insulin signaling and the anti-inflammatory effects by the chloroform fraction of ethanolic extract of Nymphaea rubra flowers in TNF-alpha-induced insulin resistance in the rat skeletal muscle cell line (L6 myotubes) to dissect out its anti-hyperglycemic mechanism. N. rubra enhances the GLUT4-mediated glucose transport in a dose dependent manner and also increases the tyrosine phosphorylation of both IR-beta and IRS-1, and the IRS-1 associated PI-3 kinase activity in TNF-alpha-treated L6 myotubes. Moreover, N. rubra decreases Ser(307) phosphorylation of IRS-1 by the suppression of JNK and NF-kappa B activation. In conclusion, N. rubra reverses the insulin resistance by the inhibition of c-Jun NH2-Terminal Kinase and Nuclear-kappa B.