Monocytes can be differentiated into macrophages in vivo and these cells play an important role in innate and adaptive immune responses. To reveal the global gene transcription change that occurs during monocyte to macrophage differentiation, we performed genome-wide RNA sequencing and analyses in human primary monocytes and monocyte-derived macrophages. We show that 1208 genes (with >twofold differences) were differentially expressed in macrophages compared with monocytes, including 800 upregulated and 408 downregulated genes. Gene ontology, pathway, and protein-protein interaction analyses indicated that the upregulated genes were related to macrophage functions in phagocytosis, metabolic processes, and cell cycle. The majority of downregulated genes comprised genes involved in the inflammatory response and locomotion. Genes encoding transcription regulatory factors, such as FOXO1, RUNX3, NF-κB1, and C/EBP δ, were highly expressed in monocytes and appeared to function in significant transcriptional repression, resulting in slight metabolic activity. Our transcriptome comparison between human monocytes and monocyte-derived macrophages using RNA sequencing revealed novel molecules and pathways associated with the differentiation process. These molecules and pathways may represent candidate targets involved in the pathophysiology of these important immune cells.