Abstract Analysis and interpretation of RNA-Seq data remain a challenge for bench scientists. We aim to develop a user-friendly web application for exploratory data analysis (EDA), differential expression, and pathway analysis using summarized gene-level data from RNA-Seq, DNA microarray or other platforms. The key idea of iDEP (integrated Differential Expression and Pathway analysis) is to make many powerful R/Bioconductor packages easily accessible by wrapping them under a graphical interface, alongside annotation databases. For EDA, it performs hierarchical clustering, k-means clustering, and principal component analysis. iDEP detects differentially expressed genes using the limma and DESeq2 packages. For a group of co-expressed genes, it identifies enriched gene ontology (GO) terms as well as transcription factor binding motifs in promoters. Pathway analysis can be performed using packages like GAGE, GSEA, PGSEA, or ReactomePA. iDEP can also detect chromosomal gain or loss using the PREDA package. iDEP uses annotation of 163 metazoa and 45 plant genomes in Ensembl for ID mapping and GO functional categorization. Common gene IDs including microarray probe names can be automatically recognized. Pathway information was also compiled from databases like KEGG, Reactome, MSigDB, GSKB, GeneSetDB, and araPath. As an example, we analyzed an RNA-Seq dataset involving siRNA-mediated Hoxa1 knockdown in lung fibroblasts, and identified the downregulation of cell-cycle genes, in agreement with previous studies. Our analyses also reveal the possible roles of E2F1 and its target genes, including microRNAs, in blocking G1/S transition, and the upregulation of genes related to cytokines, lysosome, and neuronal parts. iDEP enables users to conduct in-depth bioinformatics analysis of transcriptomic data through a graphical interface. Freely available at http://gelab.org/idep/ Citation Format: Steven X. Ge. iDEP: integrated differential expression and pathway analysis of RNA-Seq data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-026.