Background Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process. Methods New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results. Results Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia. Conclusions Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.