The genome of diplonemid Paradiplonema papillatum, like many other protist organisms, encodes a vast swathe of hypothetical proteins, for which no annotation is currently available, and confounds interpretations that can be drawn from molecular analysis. As a result, many outstanding questions remain for this clade of heterotrophic unicellular flagellates, including their evolutionary divergence and modes of nutrition. To rectify this, we initiated subcellular proteomics on P. papillatum to resolve localization for approximately 5,000 proteins of this model diplonemid. We ultimately predict 2,435 proteins to 22 cellular compartments of this organism, for which we performed additional validation via a series of endogenous tagging procedures across new predictions. For the first time, we resolve the cell membrane of P. papillatum, showing an accumulation of previously identified carbohydrate active enzymes. Our topological reconstructions for these enzymes suggest exposure to extracellular space and supports recent proposals for the breakdown of plant and algal cell wall carbohydrates in the world’s oceans as a means of nutrition. In turn we localize various metabolic enzymes of the cell, demonstrating broad amino acid processing capabilities within the mitochondrion, along with new glycosomal-localized carbohydrate enzymes, which strengthen claims for ancestral compartmentalisation for certain steps within the modified peroxisomes shared between sister-clades kinetoplastids and diplonemids. This dataset constitutes a proteomic resource from which to explore the unique cell biology of diplonemids and clarify developmental trends throughout their evolutionary differentiation.