The myometrium undergoes substantial remodelling at the time of labour including rearrangement of the cellular contractile machinery. The regulation of this process in human myometrium at the time of labour is poorly defined but evidence in other muscles types suggests modulation by small heat shock proteins (sHSP). The aim of this study was to investigate if similar changes in sHSP occur in the myometrium at labour. Using a quantitative proteomic approach (2D-DIGE) we found a 69% decrease in the small HSP, B-crystallin, in the myometrium at labour plus multiple isoforms of HSP27. Immunoblotting using phospho-specific HSP27 antibodies (HSP27-serine15, 78 and 82) detected marked changes in HSP27 phosphorylation at labour. While total HSP27 levels were unchanged, HSP27-ser15 was 3-fold higher at labour. Co-immunoprecipitation studies showed that HSP27 co-precipitates with B-crystallin and also smooth muscle -actin. Co-immunoflorescence studies demonstrated a relocation of HSP27 from the perinuclear region to the actin cytoskeleton at labour. The functional significance of these changes was demonstrated in vitro where myometrial strips stimulated to contract with oxytocin exhibited increased HSP27-ser15 phosphorylation. Our findings provide data consistent with a novel pathway regulating human myometrial contraction at labour and identify HSP27 and B-crystallin as potential targets for future tocolytic design.