Reversible protein phosphorylation catalyzed by protein kinases and phosphatases is a well- established post-translational modification that plays a prominent role in controlling protein function and mediating cellular signals. In Arabidopsis thaliana, Shewanella-like protein phosphatase 2 (AtSLP2) is a novel bacteria-like protein phosphatase that is targeted to the mitochondrial intermembrane space (IMS) where it interacts with the mitochondrial oxidoreductase import and assembly protein 40 (MIA40). This interaction with MIA40 results in intramolecular disulfide bonds on AtSLP2 that activate its phosphatase activity. A quantitative phosphoproteomics study using AtSLP2 knockout and protein over-expression plant lines was previously able to identify TOM9 (translocase of the outer mitochondrial membrane) and PHB2 (prohibitin 2) as putative substrates of AtSLP2. The focus of the research presented here was to characterize AtSLP2’s relationship with its putative substrates in regulating mitochondrial processes from the IMS. Here, initial steps were taken in employing phosphospecific antibodies against AtSLP2’s putative substrates with immunoblots aligning with previous phosphoproteomics results that PHB2 is hyperphosphorylated in the absence of AtSLP2 and conversely hypophosphorylated in its over-expression. Additionally, activity assays using phosphorylated peptides revealed that AtSLP2 has a clear preference towards substrates with more acidic motifs. To explore the influence of AtSLP2’s activity on different mitochondrial functions, a quantitative proteomics study was undertaken to compare perturbations in protein abundance from wild type, AtSLP2 knockout and protein over-expression plant lines. The findings from this study suggests that AtSLP2 plays an important role in regulating mitochondrial energy metabolism and branched-chain amino acid metabolism. Lastly, we tested the AtSLP2 model generated by AlphaFold to review the predicted disulfide bonds catalyzed by MIA40. Using site-directediimutagenesis and subsequent phosphatase assays, we substituted multiple highly conserved cysteine residues within AtSLP2, but were unable to confirm which cysteines are involved in disulfide bond formation. In summary, the results presented here provide a biochemical, structural, and functional characterization of a novel mitochondrial protein phosphatase in plants.