Endometrial cysteine-rich protein 61 (CYR61, CCN1) is a growth factor-inducible gene whose expression is elevated during the proliferative phase of the menstrual cycle and which has been implicated in the pathogenesis of endometriosis. This study aimed to define the mediators of epidermal growth factor (EGF) signalling on CYR61 expression in spontaneously immortalised human endometrial epithelial cells (HES) as a model system. After 30 min of EGF treatment, the receptor was phosphorylated and internalised as well as mRNA CYR61 increased in HES cells. However, neither inhibition of C-terminal EGF receptor (EGFR)-phosphorylation nor blockage of the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was able to reduce CYR61 levels. Surprisingly, the HES cells showed upregulation of CYR61 mRNA expression after inhibition of the MAPK/ERK pathway when treated with EGF. Specific inhibitor studies identified the contribution of Janus kinase 2 (JAK2) and the signal transducer and activator of transcription protein STAT3 to the regulation of CYR61 expression. The JAK2/STAT3 interaction contributed to the basal expression of CYR61 and mediated EGF-driven regulation of CYR61 after 30 and 120 min of treatment. In summary, EGF-mediated CYR61 upregulation in HES cells involves STAT3 and is counter-regulated by the EGFR/MAPK/ERK pathway.