The precise molecular mechanisms controlling progesterone receptor (PR)-mediated gene regulation within the human myometrium in pregnancy and in labour remain poorly defined. PR recruit different nuclear co-activators/co-repressors to mediate receptor-specific transcription regulation and expression of PR, and these co-factors may alter within the myometrium during pregnancy and labour. The aims of this study were to test the hypotheses that i) the human splicing and transcription factor, polypyrimidine tract binding protein-associated splicing factor (PSF), is spatially and temporally regulated in the myometrium during pregnancy and labour; ii) PSF influences the expression of myometrial PR and iii) the action of PR in regulating specific hormone response target genes in the human myometrium may involve PSF. Immunoblotting indicated that PSF expression is significantly up-regulated within the human myometrium as pregnancy progresses, in particular within the upper uterine region, and levels remain elevated in labour. Co-immunoprecipitations and DNA-binding assays show that PSF directly interacts with nuclear PR and glucocorticoid receptor (GR) and specific co-regulatory proteins, all of which have defined roles as co-activators or co-repressors in gene regulation. Over-expression and inhibition of PSF by transient transfection and RNAi respectively alters expression of myometrial PR and GR and may influence expression of two PR/GR-target genes, cyclooxygenase-2 and histone deacetylase-2. These findings are suggestive of a role for myometrial PSF as a nuclear co-regulator in the regulation of specific hormone receptor genes and their target hormone response genes.