© 2015 by the Endocrine Society. Islet-1 (Isl1) is a Lin11, Isl1, Mec3 (LIM)-homeodomain transcription factor important for pancreatic islet cell development, maturation, and function, which largely requires interaction with the LIM domain-binding protein 1 (Ldb1) coregulator. In other tissues, Ldb1 and Isl1 interact with additional factors to mediate target gene transcription, yet few protein partners are known in β-cells. Therefore, we hypothesize that Ldb1 and Isl1 participate in larger regulatory complexes to impact β-cell gene expression. Totest this, we used cross-linked immunoprecipitation and mass spectrometry to identify interacting proteins from mouse β-cells. Proteomic datasets revealed numerous interacting candidates, including a member of the single-stranded DNA-binding protein (SSBP) coregulator family, SSBP3. SSBPs potentiate LIM transcription factor complex activity and stability in other tissues. However, nothing was known of SSBP3 interaction, expression, or activity in β-cells. Our analyses confirmed that SSBP3 interacts with Ldb1 and Isl1 in β-cell lines and in mouse and human islets and demonstrated SSBP3 coexpression with Ldb1 and Isl1 pancreas tissue. Furthermore, β-cell line SSBP3 knockdown imparted mRNA deficiencies similar to those observed upon Ldb1 reduction in vitro or in vivo. This appears to be (at least) due to SSBP3 occupancy of known Ldb1-Isl1 target promoters, including MafA and Giplr. This study collectively demonstrates that SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes, required for expression of critical β-cell target genes.