Bit1 (Bcl-2 inhibitor of transcription) is a mitochondrial protein that induces caspase-independent apoptosis upon its release into the cytoplasm. Bit1 is primarily associated with anoikis (cell death induced by detachment from the extracellular matrix), because the apoptotic function of Bit1 is inhibited by integrin-mediated cell attachment but not by many other antiapoptotic treatments. Here, we show that protein kinase D (PKD) regulates Bit1 apoptotic function. Overexpression of constitutively active PKD or PKD activation by treatment with phorbol 12-myristate 13-acetate results in phosphorylation of two serine residues (Ser5 and Ser87) in a form of Bit1 that is confined to the cytoplasm and concomitantly increases the apoptotic activity of cytoplasmic Bit1. Conversely, suppressing PKD activity with pharmacological inhibitors or small interfering RNA approaches attenuates apoptosis induced by cytoplasmic Bit1. Furthermore, PKD regulates induction of cell death by wild-type Bit1 following loss of cell attachment to the extracellular matrix. Activation of PKD enhances Bit1 function in anoikis, whereas inhibiting PKD function with pharmacological inhibitors or small interfering RNA compromises the ability of Bit1 to induce anoikis. The induction of Bit1-mediated apoptosis by PKD is in part attributable to the release of Bit1 from mitochondria to the cytoplasm as a consequence of phosphorylation of Ser5 in the mitochondrial localization sequence of Bit1. Consistent with the regulatory role of PKD in the anoikis function of Bit1, we found that cell attachment to fibronectin inhibits PKD activity. These studies identify the PKD serine/threonine kinase as one of the signaling molecules through which integrin-mediated cell attachment controls Bit1 activity and anoikis.