Breakdown of serine by the enzyme serine hydroxymethyltransferase (SHMT) produces glycine and one-carbon (1C) units. These serine catabolites provide important metabolic intermediates for the synthesis of nucleotides, as well as methyl groups for biosynthetic and regulatory methylation reactions. Recently, it has been shown that serine catabolism is required for efficient cellular respiration. Using CRISPR-Cas9 gene editing, we demonstrate that the mitochondrial SHMT enzyme, SHMT2, is essential to maintain cellular respiration, the main process through which mammalian cells acquire energy. We show that SHMT2 is required for the assembly of Complex I of the respiratory chain. Furthermore, supplementation of formate, a bona fide 1C donor, restores Complex I assembly in the absence of SHMT2. Thus, provision of 1C units by mitochondrial serine catabolism is critical for cellular respiration, at least in part by influencing the assembly of the respiratory apparatus.