Cristae membrane state plays a central role in regulating mitochondrial function and cellular metabolism. The protein Optic atrophy 1 (Opa1) is an important crista remodeler that exists as two forms in the mitochondrion, a membrane-anchored long form (l-Opa1) and a processed short form (s-Opa1). The mechanisms for how Opa1 influences cristae shape have remained unclear due to lack of native three-dimensional views of cristae. We perform in situ cryo-electron tomography of cryo-focused ion beam milled mouse embryonic fibroblasts with defined Opa1 states to understand how each form of Opa1 influences cristae architecture. In our tomograms, we observe a variety of cristae shapes with distinct trends dependent on s-Opa1:l-Opa1 balance. Increased l-Opa1 levels promote cristae stacking and elongated mitochondria, while increased s-Opa1 levels correlated with irregular cristae packing and round mitochondria shape. Functional assays indicate a role for l-Opa1 in wild-type apoptotic and calcium handling responses, and show a compromised respiratory function under Opa1 imbalance. In summary, we provide three-dimensional visualization of cristae architecture to reveal relationships between mitochondrial ultrastructure and cellular function dependent on Opa1-mediated membrane remodeling.|Mitochondrial inner-membrane morphology plays essential roles in regulating organelle physiology, but how molecular factors regulate cristae form and function is unclear. In this article, in situ cryo-electron tomography coupled to functional experiments show how different variants of Opa1 influence mitochondrial membrane architecture and activities.Mitochondria with predominantly membrane-anchored long Opa1 isoform (l-Opa1) show crista stacking, longer cristae, reduced globular cristae and an absence of tubular cristae. Mitochondria with mostly processed short Opa1 isoform (s-Opa1) showed irregular cristae packing with wider cristae junctions and narrower cristae. l-Opa1 expressing cells show wild-type-like cristae junction properties, apoptotic response and calcium handling. Imbalance in Opa1 processing leads to compromised respiratory function and an increase in amorphous cristae.|In situ cryo-electron tomography of mitochondria reveals the role of balanced Opa1 proteolytic processing in the determination of cristae structure.