AbstractChromatin modifiers and transcriptional cofactors (collectively referred to as CFs) work with DNA-binding transcription factors (TFs) to regulate gene expression. In multicellular eukaryotes, distinct tissues each execute their own gene expression program for accurate differentiation and subsequent functionality. While the function of TFs in differential gene expression has been studied in detail in many systems, the contribution of CFs has remained less explored. Here, we uncovered the contributions of CFs to gene regulation in theCaenorhabditis elegansintestine. We first annotated 366 CFs encoded by theC. elegansgenome and assembled a library of 335 RNAi clones. Using this library, we analyzed the effects of individually depleting these CFs on the expression of 19 fluorescent transcriptional reporters in the intestine and identified 216 regulatory interactions. We found that different CFs regulate different promoters, and that both essential and intestinally expressed CFs have the greatest effects on promoter activity. We did not find all members of CF complexes acting on the same set of reporters but instead found diversity in the promoter targets of each complex component. Finally, we found that previously identified activation mechanisms for theacdh-1promoter use different CFs and TFs. Overall, we demonstrate that CFs function specifically rather than ubiquitously at intestinal promoters and provide an RNAi resource for reverse genetic screens.