In pituitary gonadotrophs, GnRH induces expression of the mitogen-activated protein kinases (MAPK3/1) dephosphorylating enzyme, dual-specificity phosphatase 1 (DUSP1). Here we examined DUSP1 expression levels following pulsatile GnRH stimulation of the LbetaT2 gonadotroph cells. DUSP1 expression was increased more prominently following high-frequency (every 30 min) GnRH pulse stimulation (7.02- ± 1.47-fold) than low-frequency (every 120 min) GnRH pulses (2.68- ± 0.09-fold). With high-frequency GnRH pulses, DUSP1 expression increased by 2.89- ± 0.32-fold 2 h after GnRH pulse initiation (four 5-min pulses). DUSP1 expression was not induced following lower frequency GnRH pulses, even when the GnRH concentration was increased. Under high-frequency conditions, MAPK3/1 phosphorylation was observed 10 min after the GnRH pulse and decreased to basal levels after 25 min. However, MAPK3/1 dephosphorylation did not occur concurrently with DUSP1 expression. Overexpression of MAP3K1, a kinase upstream of MAPK3/1, increased both the Lhb and the Fshb subunit promoter activities, which could be completely inhibited by cotransfection with DUSP1-expressing vectors. Serum response factor (Srf) promoter activities induced by MAP3K1 were also prevented by DUSP1 overexpression, confirming that MAPK3/1 has an important role in gonadotropin subunit gene expression. Both high- and low-frequency GnRH pulse stimulation failed to increase the Lhb and Fshb subunit gonadotropin gene expression levels upon DUSP1 overexpression. Our study demonstrates that DUSP1 is specifically expressed following high-frequency GnRH pulses and that this effect may participate in the differential regulation of gonadotropin subunit expression in association with MAPK3/1 phosphorylation.