Tomato spotted wilt virus (TSWV), a member of the Tospovirus genus within the Bunyaviridae, is an economically important plant pathogen with a worldwide distribution. TSWV is transmitted to plants via thrips (Thysanoptera: Thripidae), which transmit the virus in a persistent propagative manner. The envelope glycoproteins, G(N) and G(C), are critical for the infection of thrips, but they are not required for the initial infection of plants. Thus, it is assumed that the envelope glycoproteins play important roles in the entry of TSWV into the insect midgut, the first site of infection. To directly test the hypothesis that G(N) plays a role in TSWV acquisition by thrips, we expressed and purified a soluble, recombinant form of the G(N) protein (G(N)-S). The expression of G(N)-S allowed us to examine the function of G(N) in the absence of other viral proteins. We detected specific binding to thrips midguts when purified G(N)-S was fed to thrips in an in vivo binding assay. The TSWV nucleocapsid protein and human cytomegalovirus glycoprotein B did not bind to thrips midguts, indicating that the G(N)-S-thrips midgut interaction is specific. TSWV acquisition inhibition assays revealed that thrips that were concomitantly fed purified TSWV and G(N)-S had reduced amounts of virus in their midguts compared to thrips that were fed TSWV only. Our findings that G(N)-S binds to larval thrips guts and decreases TSWV acquisition provide evidence that G(N) may serve as a viral ligand that mediates the attachment of TSWV to receptors displayed on the epithelial cells of the thrips midgut.