The Spsb family of genes encode well-conserved proteins of unknown function. Mammalian Spsb genes are likely the result of three separate duplication and divergence events during vertebrate evolution. The phylogenetic relationship along with expression and regulation of Spsb genes may offer insight into the evolution and function of this gene family in vertebrates. We have established that Spsb genes are expressed in numerous tissues, however their pattern and level of expression is tissue-dependent. Further, only Spsb1 is responsive to stress caused by ethanol exposure in the mouse brain, which suggests that Spsb genes have acquired different regulatory mechanisms. Analysis of cis-regulatory elements supports this, but also reveals some common regulatory modules involved in cell proliferation and stress response. Our results contribute to the growing body of data on the expression and function of Spsb genes, which serve as a model for studies on the origin, divergence and specialization of eukaryotic gene families.