Cytochrome b 559 (Cyt b 559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b 559 remains unclear. Cyt b 559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b 559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b 559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b 559 and implications for revealing the physiological functions of Cyt b 559 in PSII.