Poor sleep hygiene is a growing problem, with detrimental effects on many biological systems. The pituitary gland plays a crucial role in the regulation of sleep and the stress response, and its dysfunction leads to sleep-related disorders. However, the interaction between these critical functions remains unclear. Thus, we performed a comparative, whole-transcriptome, analysis to identify stress-induced genes and relevant pathways that may be affected by sleep deprivation. One day following 12 h of Paradoxical Sleep Deprivation (PSD), mice were restrained for 20 min. Gene expression changes in the pituitary were assessed via RNA-Seq and Gene Ontology in PSD and/or restrained groups compared to controls. We show that restraint triggers transcriptional responses involved in hormone secretion, the glucocorticoid response, and apoptosis in both sexes, with 285 differentially expressed genes in females and 93 in males. When PSD preceded restraint stress, the numbers of differentially expressed genes increased to 613 in females and 580 in males. The pituitary transcriptome of restraint+PSD animals was enriched for microglia and macrophage proliferation, cellular response to corticosteroids, and apoptosis, among others. Finally, we identify sex-specific differences in restraint-induced genes following PSD. These findings provide genetic targets to consider when studying sleep and the response to stress.