The US FDA approval of broad-spectrum histone deacetylase (HDAC) inhibitors has firmly laid the cancer community to explore HDAC inhibition as a therapeutic approach for cancer treatment. Hitting one HDAC member could yield clinical benefit but this required a complete understanding of the functions of the different HDAC members. Here we explored the consequences of specific HDAC5 inhibition in cancer cells. We demonstrated that HDAC5 inhibition induces an iron-dependent reactive oxygen species (ROS) production, ultimately leading to apoptotic cell death as well as mechanisms of mitochondria quality control (mitophagy and mitobiogenesis). Interestingly, adaptation of HDAC5-depleted cells to oxidative stress passes through reprogramming of metabolic pathways towards glucose and glutamine. Therefore, interference with both glucose and glutamine supply in HDAC5-inhibited cancer cells significantly increases apoptotic cell death and reduces tumour growth in vivo; providing insight into a valuable clinical strategy combining the selective inhibition of HDAC5 with various inhibitors of metabolism as a new therapy to kill cancer cells.