Cancer cells, including malignant lymphoma cells, alter their metabolism, termed "metabolic reprograming," on initiation of malignant transformation as well as upon accumulation of genetic abnormalities. Here, to identify a novel therapeutic target involved in the metabolic changes during malignant lymphoma, we performed global analyses combined with shotgun proteomics, in silico database analysis, and clinic-pathologic analysis of nonneoplastic lymphoid tissue and malignant lymphoma tissue and verified the molecular functions in vitro. In total, 2002 proteins were detected from both samples and proteins related to fatty acid beta-oxidation (FAO) were detected more frequently in malignant lymphoma tissue. Consequently, the most frequently detected protein, the mitochondrial trifunctional enzyme subunit-alpha (HADHA), was identified as a potential target. Immunohistochemical analyses revealed that HADHA tended to be overexpressed in a high-grade subtype of malignant lymphoma tissue. Clinicopathologic study revealed that HADHA overexpression was correlated with significantly worse overall survival (P = 0.013) and was an independent prognostic predictor in diffuse large B-cell lymphoma (P = 0.027). In vitro, downregulation of HADHA negatively regulated cell growth by causing G0/G1 arrest (P = 0.0008) similar to treatment with etomoxir, an inhibitor of FAO (P = 0.032). Moreover, downregulation of HADHA increased the susceptibility to doxorubicin (P = 0.002) and etoposide (P = 0.004). Moreover, these phenotypes were confirmed in an HADHA knockout system. Thus, we provide a basis for a novel therapeutic strategy through the regulation of HADHA and FAO in patients with refractory malignant lymphoma.