Pituitary tumor cells have a poor response to the growth inhibitory effect of TGFβ1, possibly resulting from the cross talk of TGFβ/Smads signal with other signaling pathways, an undescribed mechanism in these tumoral cells. To address this hypothesis, we investigated whether the mitogen-activated extracellular signal-regulated kinase (MEK)/ERK1/2 and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathways were able to regulate the antimitogenic effect of TGFβ1 on GH3B6 cells. TGFβ1 treatment decreased the cell proliferation and induced an activation of mothers against decapentaplegic homolog 2/3 (Smad2/3), effects that were potentiated by MEK and PI3K inhibitors, thus indicating the existence of a cross talk between TGFβ1/Smad with the MEK/ERK1/2 or PI3K/Akt pathways. In addition, through immunoprecipitation assays, a direct interaction was observed between Smad2/3-ERK1/2 and Smad2/3-Akt, which decreased when the GH3B6 cells were incubated with TGFβ1 in the presence of MEK or PI3K inhibitors, thereby suggesting that the ERK1/2- and Akt-activated states were involved. These Smad2/3-ERK1/2 and Smad2/3-Akt associations were also confirmed by confocal and transmission electron microscopy. These findings indicate that the TGFβ1-antimitogenic effect in GH3B6 cells was attenuated by the MEK/ERK1/2 and PI3K/Akt pathways via modulating Smad2/3 phosphorylation. This molecular mechanism could explain in part the refractory behavior of pituitary tumor cells to the inhibitory effect of TGFβ1.