The salivary system in mammals is comprised of three independently developed pairs of organs, the parotid, submaxillar, and sublingual glands. Each gland is composed of various ductal and acinar cell types that fulfill multiple roles. However, the molecular mechanisms regulating their biogenesis and functions are still largely unknown. In this paper, we report that two class B basic helix-loop-helix (bHLH) transcriptional regulators delineate the ductal and the acinar cells in salivary glands. Sgn1, a novel class B bHLH factor, is specifically expressed in the salivary duct cells, while the acinar cells are characterized by the expression of another class B bHLH factor, Mist1. The molecular nature of Sgn1 was also investigated: it binds to specific sequences of DNA as a dimer with a class A bHLH factor and acts as a negative transcriptional regulator against other bHLH factors. This study provides an important cue towards better understanding of the generation and function of multiple cell types in salivary glands. In addition, Sgn1 expression exhibits a reverse relationship with the development of male phenotypes, suggesting its role in gender dimorphism in the salivary glands.