In mammals, the functional significance of the presence of evolutionarily conserved, multiple non-allelic H1 variants remains unclear. We used a unique overproduction approach coupled with cell cycle synchronization and early time point assays to assess differential effects of H1 variants, H1c and H1(0), on global gene expression in the absence of compensatory events that may mask variant-specific effects. We found that H1c and H1(0) act primarily as specific rather than global regulators of gene expression. Many of the genes affected were uniquely targeted by either H1c or H1(0), affirming that H1 variants have some unique roles. We also identified genes that were affected by both variants, in which cases the expression of these genes was, for the most part, affected similarly by both the variants. This observation suggests that as well as having specific functions, the H1 variants share common roles in the organization of chromatin. We further noted that H1(0) repressed more genes than did H1c, which may underlie the prevailing notion that H1(0) is a stronger repressor of transcription.