We previously reported the Bcl-2/Bcl-xL-bispecific activity of the 2'-O-(2-methoxy)ethyl (2'-MOE)-modified gapmer antisense oligonucleotide 4625. This oligonucleotide has 100% complementarity to Bcl-2 and three mismatches to Bcl-xL. In the present study, the isosequential locked nucleic acid (LNA)-modified oligonucleotide 5005 was generated, and its ability to further improve the downregulation of the two antiapoptotic targets in tumor cells was examined. We demonstrate that compared with 4625, 5005 more effectively decreased the expression of the mismatching Bcl-xL target gene in MDA-MB-231 breast and H125 lung cancer cells. In both cell lines, antisense activity caused decreased cell viability by induction of apoptosis. Moreover, in combination with various anticancer agents, 5005 reduced tumor cell viability more effectively than 4625. We describe for the first time the functional comparison of isosequential Bcl-2/Bcl-xL-bispecific 2'-MOE and LNA-modified antisense oligonucleotides and report that the LNA analog more effectively downregulated the two apoptosis inhibitors overexpressed in human tumors. Our data underscore the ability of LNA modifications to enhance the efficacy and favorably modulate the target specificity of antisense oligonucleotides.