Syntaxin 10 is a soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein localized to the trans-Golgi network (TGN), where two other members of the syntaxin family, syntaxins 6 and 16, also reside. The role of syntaxin 10 in regulating TGN protein traffic is not yet defined. Syntaxin 10 co-localizes well with syntaxins 6 and 16 at the TGN in interphase cells, and appears to interact with both syntaxin 6 and 16 as evidenced by co-immunoprecipitation analyses. However, unlike syntaxin 6 and 16, neither syntaxin 10 antibodies nor its cytosolic domain inhibits endosome-TGN transport of shiga toxin. Syntaxin 16 knockdown with small interfering RNA (siRNA) affects the TGN localization of syntaxin 6 but not syntaxin 10, and clearly inhibits endosome-TGN transport. On the other hand, knockdown of syntaxin 10 expressions had no significant effect on the TGN localization of syntaxin 6 and 16, and did not inhibit endosome-TGN transport. Unlike syntaxin 16, syntaxin 10 does not interact specifically with Vps45, the Sec1/Munc18 (SM) family member at the TGN. On the other hand, syntaxin 10 reciprocally co-immunoprecipitated endosomal syntaxin 12/13, and knockdown of syntaxin 10 expressions affects the surface expression of transferrin receptor (TfR) and seems to induce the formation of an immobile TfR pool. Therefore, in spite of its co-localization and possible interaction with syntaxin 16, syntaxin 10 is not part of the syntaxin 16-based SNARE complex involved in endosome-TGN transport, and may have a hitherto unrecognized function in the TGN-endosome boundary.