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Proteins in the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3) family are involved in the regulation of many intracellular processes. We have examined the interaction of 14-3-3 with peptidylarginine deiminase, type VI (PADI6), an abundant protein found in mammalian oocytes, eggs and early embryos. Peptidylarginine deiminases catalyze the posttranslational modification of peptidylarginine to citrulline. PADI6 is associated with oocyte cytoplasmic sheets containing intermediate filaments and it has been shown that PADI6-deficient mice are infertile due to disruption of development beyond the two-cell stage. We found that PADI6 undergoes a dramatic developmental change in phosphorylation during oocyte maturation. This change in phosphorylation is linked to an interaction of PADI6 with14-3-3 in the mature egg. Recombinant glutathione-s-transferase (GST) 14-3-3 pull-down experiments and transgenic tandem affinity purification with liquid chromatography/mass spectrometry demonstrates a binding interaction between 14-3-3 and PADI6 in mature eggs, but not in the immature oocytes. 14-3-3 proteins modulate or complement intracellular events involving phosphorylation dependent switching or protein modification. These results indicate that phosphorylation and/or 14-3-3 binding may serve as a means of intracellular PADI6 regulation.