Nutritional systems biology is an evolving research field aimed at understanding nutritional processes at a systems level. It is known that the development of cancer can be influenced by the nutritional status, and the link between vitamin D status and different cancer types is widely investigated. In this study, we performed an integrative network-based analysis using a publicly available data set studying the role of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in prostate cancer cells on mRNA and microRNA level. Pathway analysis revealed 15 significantly altered pathways: eight more general mostly cell cycle-related pathways and seven cancer-specific pathways. The changes in the G1-to-S cell cycle pathway showed that 1,25(OH)2D3 down-regulates the genes influencing the G1-to-S phase transition. Moreover, after 1,25(OH)2D3 treatment the gene expression in several cancer-related processes was down-regulated. The more general pathways were merged into one network and then extended with known protein-protein and transcription factor-gene interactions. Network algorithms were used to (1) identify active network modules and (2) integrate microRNA regulation in the network. Adding microRNA regulation to the network enabled the identification of gene targets of significantly expressed microRNAs after 1,25(OH)2D3 treatment. Six of the nine differentially expressed microRNAs target genes in the extended network, including CLSPN, an important checkpoint regulator in the cell cycle that was down-regulated, and FZD5, a receptor for Wnt proteins that was up-regulated. The extendable network-based tools PathVisio and Cytoscape enable straightforward, in-depth and integrative analysis of mRNA and microRNA expression data in 1,25(OH)2D3-treated cancer cells.