One of the most potentially promising approaches to convert renewable solar energy to hydrogen biofuel is through photosynthetic water splitting by Cyanobacteria. Efficient and economical bioconversion of sunlight requires comprehensive understanding and optimization of the biological processes. Synechocystis sp. PCC 6803 evolves hydrogen via a bidirectional hydrogenase in the dark under anoxic conditions. Our work seeks to investigate and optimize metabolic and regulatory interactions between hydrogenase regulation and hydrogen production, photosynthesis and respiration, and nutrient status. We have designed and validated whole-genome oligonucleotide (70-mer) microarray as a platform to probe the genome-wide transcriptional regulation under macronutrient limiting conditions such as CO₂, nitrogen, phosphorus and sulfur. As a first step the physiological and transcriptional responses to sulfur deprivation by Synechocystis were investigated. Our data showed that sulfur deprivation conditions down-regulate photosystems, Cytochrome b6/f complement and ATP generation, while sulfate transport, phycobilisome degradation protein genes, along with many other genes of unknown function, were up-regulated. This transcriptional reprogramming was confirmed not due to a growth-stage related response, but sulfur element starvation which significantly improved hydrogen production. The metabolic and regulatory information derived from this study will be used for improving the hydrogen productivity and sustainability by Cyanobacteria.