Additional sex comb-like 1 (ASXL1, 170 kDa), a mammalian homolog of Drosophila ASX, was identified as a protein that interacts with RAR in the presence of retinoic acid (RA). Systematic binding assays showed that the C-terminal nuclear receptor (NR) box (LVMQLL) of ASXL1 and the AF-2 AD core of RAR are critical for ligand-dependent interaction. The interaction was confirmed using in vitro GST pull-down and in vivo immunoprecipitation (IP) assays. Confocal microscopy revealed that ASXL1 localizes in the nucleus. In addition to the intrinsic transactivation function of ASXL1, its cotransfection together with an RA-responsive luciferase reporter increased the RAR activity. This ASXL1 activity appears to be mediated through the functional cooperation with SRC-1, as shown by GST pull-down, IP, chromatin IP, and transcription assays. In the presence of ASXL1, more acetylated histone H3 was accumulated on the RA-responsive promoter in response to RA. Finally, stable expression of ASXL1 increased the expression of endogenous RA-regulated genes, and enhanced the antiproliferative potential of RA. Overall, these results suggest that ASXL1 is a novel coactivator of RAR that cooperates with SRC-1, and implicates it as a potential antitumor target of RA in RA-resistant cancer cells.