Succinate dehydrogenase (SDH) plays pivotal roles in maintaining cellular metabolism, modulating regulatory control over both the tricarboxylic acid cycle and oxidative phosphorylation to facilitate energy production within mitochondria. Given that SDH malfunction may serve as a hallmark triggering pseudo-hypoxia signaling and promoting tumorigenesis, elucidating the impact of SDH assembly defects on mitochondrial functions and cellular responses is of paramount importance. In this study, we aim to clarify the role of SDHAF2, one assembly factor of SDH, in mitochondrial respiratory activities. To achieve this, we utilize the CRISPR/Cas9 system to generate SDHAF2 knockout in HeLa cells and examine mitochondrial respiratory functions. Our findings demonstrate a substantial reduction in oxygen consumption rate in SDHAF2 knockout cells, akin to cells with inhibited SDH activity. In addition, in our in-gel activity assays reveal a significant decrease not only in SDH activity but also in cytochrome c oxidase (COX) activity in SDHAF2 knockout cells. The reduced COX activity is attributed to the assembly defect and remains independent of SDH inactivation or SDH complex disassembly. Together, our results indicate a critical role of SDHAF2 in regulating respiration by facilitating the assembly of COX.