The progesterone receptor (PR) plays important roles in the establishment and maintenance of pregnancy. By dynamic interactions with co-regulators, PR represses the expression of genes that increase the contractile activity of myometrium and contribute to the initiation of labor. We have previously shown that PSF can function as a PR co-repressor. In this report, we demonstrated that the PSF heterodimer partner, p54nrb, can also function as a transcription co-repressor, independent of PSF. p54nrb interacts directly with PR independent of progesterone. In contrast to PSF, p54nrb does not enhance PR protein degradation nor block PR binding to DNA. Rather, p54nrb recruits mSin3A through its N-terminus to the PR/DNA complex, resulting in an inhibition of PR-mediated transactivation of the PRE-luciferase reporter gene. PR also repressed transcription of the connexin 43 gene (Gja1), an effect dependent on the presence of an AP-1 site within the proximal Gja1 promoter. Mutation of this site abolished PR-mediated repression and decreased the recruitment of PR and p54nrb onto the Gja1 promoter. Furthermore, knockdown p54nrb expression by siRNA alleviated PR-mediated repression on Gja1 transcription, while overexpression of p54nrb enhanced it. In the physiologic context of pregnancy, p54nrb protein levels decrease with the approach of labor in the rat myometrium. We conclude that p54nrb is a transcriptional co-repressor of PR. Decreased expression of p54nrb at the time of labor may act to de-repress PR-mediated inhibition on connexin 43 expression and contribute to the initiation of labor.