Human karyopherin beta3, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin beta3 complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin beta3 and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin beta3 and apo A-I. In addition, overexpression of karyopherin beta3 significantly increased apo A-I secretion. These results suggest that karyopherin beta3 plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin beta3 and coronary artery diseases associated with apo A-I.