In pediatric neuroblastoma, MYCN-amplification correlates to poor clinical outcome and new treatment options are needed for these patients. Identifying the metabolic adaptations crucial for tumor progression may be a promising strategy to discover novel therapeutic targets. Here, we have combined proteomics, gene expression profiling, functional analysis, and metabolic tracing to decipher the impact of MYCN on neuroblastoma cell metabolism. We found that high MYCN levels are correlated with altered expression of proteins involved in multiple metabolic processes, including enhanced glycolysis and increased oxidative phosphorylation. Unexpectedly, we discovered that MYCN-amplified cells showed de novo glutamine synthesis. Furthermore, inhibition of β-oxidation reduced the viability of MYCN-amplified cells in vitro and decreased tumor burden in vivo, while not affecting non-MYCN-amplified tumors. Our data provide information on metabolic processes in MYCN expressing tumors, which could be exploited for the development of novel targeted therapies.