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<p>Abstract</p> <p>Background</p> <p>Nuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known.</p> <p>Results</p> <p>A 703 bp encoding Ntf2 and a 1233 bp encoding Ran full-length cDNAs were cloned from <it>Helicoverpa armigera</it>, and named <it>Ha-Ntf2 </it>and <it>Ha-Ran</it>, respectively. Northern blot and immunoblotting revealed that <it>Ha-Ntf2 </it>had an obviously higher expression levels in the head-thorax and integument of the metamorphically committed larvae. In contrast, the expression of Ha-Ran did not show obvious variation at various developmental stages in four tissues by immunoblotting analysis, except in the midgut, which showed increased expression from 5th-36 h (molting) to 6th-48 h. Both expressions of <it>Ha-Ntf2 </it>and <it>Ha-Ran </it>could be upregulated by 20E <it>in vitro</it>. Immunohistochemistry revealed that Ha-Ntf2 and Ha-Ran were primarily localized in the nucleus of various tissues. Protein binding assay and co-immunoprecipitation indicated that Ha-Ntf2 and Ha-Ran can combine with each other <it>in vitro </it>and <it>in vivo</it>. Knock down of <it>Ha-Ntf2 </it>or <it>Ha-Ran </it>by RNAi resulted in the suppression of other 20E regulated genes including <it>EcR-B1</it>, <it>USP1</it>, <it>E75B</it>, <it>BR-CZ2</it>, <it>HHR3 </it>and <it>Ha-eIF5c</it>. In addition, the knockdown of <it>Ha-Ntf2 </it>resulted in Ha-Ran being prevented in the cytoplasm. The nuclear location of the ecdysone receptor b1 (EcR-B1) was also blocked after the knockdown of <it>Ha-Ntf2 </it>and <it>Ha-Ran</it>.</p> <p>Conclusion</p> <p>These evidences suggested that Ha-Ntf2 and Ha-Ran participated in the 20E signal transduction pathway by regulating the location of EcR-B1.</p>