Receptor-associated late transducer (RALT) acts as a negative feedback inhibitor of ErbB receptor signaling via physical interaction with ErbB. Although RALT contains a 14-3-3 binding motif (247-RSHSGP-252), little is known about the molecular basis and significance of binding to 14-3-3. Here, we report that 14-3-3 interacts with RALT in H9c2 and COS-7 cells in a Ser-250 phosphorylation-dependent manner. An in vitro kinase assay showed that RALT is a substrate for checkpoint kinase 1 (Chk1). Interaction between ectopically expressed RALT and endogenous 14-3-3 was partially suppressed by pretreatment with the Chk1 inhibitor, UCN-01. In addition, expression of constitutively active Chk1 (Chk11-365 ) resulted in increased phosphorylation of the RALT 14-3-3 binding motif and enhanced the interaction between RALT and 14-3-3θ. Furthermore, fluorescence microscopy revealed that rapid trafficking of RALT to endosome-like vesicle structures was decelerated by coexpression of Chk11-365 , whereas this coexpression had no significant impact on trafficking of the RALT S250A mutant. Finally, a cycloheximide chase assay indicated that coexpression of Chk11-365 decelerated the degradation of ectopically expressed RALT, but not that of the S250A mutant. Collectively, these results suggest that Chk1 plays a role in regulating RALT protein stability by facilitating the interaction between 14-3-3 and RALT.