Oocytes rely on the mitochondrial electron transport chain and oxidative phosphorylation to produce energy and metabolites for their growth and maturation. Mitochondrial respiration also generates potentially damaging reactive oxygen (ROS) species, in part as a by-product of complex I activity. In vitro studies indicate that long-lived human and Xenopus early-stage oocytes are insensitive to complex I inhibitors and mitigate the risk of ROS generation by excluding complex I from the respiratory chain. Here we have utilised an in vivo approach to further investigate the role of complex I activity in the function and viability of mouse oocytes during their growth and maturation. Ndufs4 is a complex I subunit which in other systems is essential for complex I formation and function. We developed an oocyte-specific Ndufs4 knockout mouse using the GDF9-Cre/LoxP system to investigate the contribution of complex I in developing oocytes in vivo. In two-month-old mutant mice, there was a minimal impact on the early-stage follicles, however, there was a significant decrease in the number of large antral follicles. In isolated fully-grown oocytes, Ndufs4 deletion markedly decreases mitochondrial membrane potential (MMP), mitochondrial ROS, mitochondrial FAD++, and ATP. Ndufs4 KO oocytes displayed delayed germinal vesicle breakdown (GVBD) and had a lower polar body extrusion (PBE) rate compared to control oocytes. Proteomics data revealed that Ndufs4 deletion markedly increased the expression of protein phosphatase Ppp1cb, providing a potential link between cell cycle defects in Ndufs4 KO oocytes. These findings demonstrate that functional complex I is critical for oocyte development and maturation.