We present RNAither, a package for the free statistical environment R which performs an analysis of high-throughput RNA interference (RNAi) knock-down experiments, generating lists of relevant genes and pathways out of raw experimental data. The library provides a quality assessment of the signal intensities, as well as a broad range of options for data normalization, different statistical tests for the identification of significant siRNAs, and a significance analysis of the biological processes involving corresponding genes. The results of the analysis are presented as a set of HTML pages. Additionally, all values and plots are available as either text files or pdf and png files.