Photoautotrophic growth in nature requires the accumulation of energy-containing molecules via photosynthesis during daylight to fuel nighttime catabolism. Many diatoms store photosynthate as the neutral lipid triacylglycerol (TAG). While the pathways of diatom fatty acid and TAG synthesis appear to be well conserved with plants, the pathways of TAG catabolism and downstream fatty acid beta-oxidation have not been characterised in diatoms. We identified a putative mitochondria-targeted, bacterial-type acyl-CoA dehydrogenase (PtMACAD1) that is present in Stramenopile and Hacrobian eukaryotes, but not found in plants, animals or fungi. Gene knockout, protein-YFP tags and physiological assays were used to determine PtMACAD1's role in the diatom Phaeodactylum tricornutum. PtMACAD1 is located in the mitochondria. Absence of PtMACAD1 led to no consumption of TAG at night and slower growth in light : dark cycles compared with wild-type. Accumulation of transcripts encoding peroxisomal-based beta-oxidation did not change in response to day : night cycles or to PtMACAD1 knockout. Mutants also hyperaccumulated TAG after the amelioration of N limitation. We conclude that diatoms utilise mitochondrial beta-oxidation; this is in stark contrast to the peroxisomal-based pathways observed in plants and green algae. We infer that this pattern is caused by retention of catabolic pathways from the host during plastid secondary endosymbiosis.