The anterior pituitary folliculostellate (FS) cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines, and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the FS TtT/GF cell line with TNF-alpha caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-alpha-evoked effects were dependent on protein phosphatase 2A (PP2A) and protein kinase C (PKC) activities. TNF-alpha did not affect total cell Cx43-PP2A catalytic subunit interaction, but it did induce PP2A catalytic subunit recruitment to the Triton X-100 insoluble subcellular fraction, in which Cx43-gap junction plaques are recovered. This recruitment temporally coincided with Cx43 phosphorylated in Ser368-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-alpha, but it did interact with the atypical PKC-zeta. Moreover, this interaction was weakened by TNF-alpha. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-alpha challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals among the FS cells.