Expression of a broad array of proteins is sexually dimorphic in rodent liver, dependent on sex-specific patterns of GH secretion. Mice carrying rsl (regulator of sex limitation) alleles, discovered as trans-acting loci affecting the mouse sex-limited protein (Slp) gene, reveal an additional axis in male-specific gene regulation. Slp expresses in adult males, but in rsl homozygous mice, Slp is also expressed in females. In this study, we examined congenic rsl strains to determine rsl's site of action, breadth of targets, and interaction with hormonal induction. We show that rsl affects Slp in liver, but not kidney, and that Rsl acts on a spectrum of male-specific liver genes, including mouse urinary proteins and a cytochrome P450 expressed predominantly by males, Cyp 2d-9, but does not act on the female-prominent P450, Cyp 2a-4. Slp expression in hypophysectomized or Tfm/Y rsl mice reveals that Rsl action is independent of GH or androgen signaling. Further, parabiosis of Rsl and rsl mice does not alter expression patterns, consistent with rsl action being liver intrinsic. Finally, Slp expression initiates earlier in rsl mice, suggesting that Rsl operates before, as well as independently of, hormonal induction. This characterization suggests Rsl functions to repress transcription of a set of genes that have in common their hormonal induction in male liver, and thus accentuates sexual dimorphism of liver gene expression.