Background Numerous genetic and genomic datasets related to complex diseases have been made available during the last decade. It is now a great challenge to assess such heterogeneous datasets to prioritize disease genes and perform follow up functional analysis and validation. Among complex disease studies, psychiatric disorders such as major depressive disorder (MDD) are especially in need of robust integrative analysis because these diseases are more complex than others, with weak genetic factors at various levels, including genetic markers, transcription (gene expression), epigenetics (methylation), protein, pathways and networks. Results In this study, we proposed a comprehensive analysis framework at the systems level and demonstrated it in MDD using a set of candidate genes that have recently been prioritized based on multiple lines of evidence including association, linkage, gene expression (both human and animal studies), regulatory pathway, and literature search. In the network analysis, we explored the topological characteristics of these genes in the context of the human interactome and compared them with two other complex diseases. The network topological features indicated that MDD is similar to schizophrenia compared to cancer. In the functional analysis, we performed the gene set enrichment analysis for both Gene Ontology categories and canonical pathways. Moreover, we proposed a unique pathway crosstalk approach to examine the dynamic interactions among biological pathways. Our pathway enrichment and crosstalk analyses revealed two unique pathway interaction modules that were significantly enriched with MDD genes. These two modules are neuro-transmission and immune system related, supporting the neuropathology hypothesis of MDD. Finally, we constructed a MDD-specific subnetwork, which recruited novel candidate genes with association signals from a major MDD GWAS dataset. Conclusions This study is the first systematic network and pathway analysis of candidate genes in MDD, providing abundant important information about gene interaction and regulation in a major psychiatric disease. The results suggest potential functional components underlying the molecular mechanisms of MDD and, thus, facilitate generation of novel hypotheses in this disease. The systems biology based strategy in this study can be applied to many other complex diseases.