In eukaryotic cells, regulated protein degradation of intracellular proteins is mediated largely by the ubiquitin proteasome system (UPS). UPS-mediated protein degradation regulates virtually all crucial aspects of cellular physiology, such as cell proliferation, cell division, cell differentiation, and cell death. Concomitantly, the deregulation by the UPS contributes to human disorders including cancer. Cellular regulation by UPS- mediated protein degradation is a highly specific and selective process that depends on time (e.g. cell cycle) and location (nucleus, mitochondria or endoplasmic reticulum). An ongoing challenge in the protein degradation field is identification of degradation signals for specific proteins that trigger their degradation by the proteasome. More than 25 years ago, the first degradation signal was discovered and defined as destabilizing N-terminal amino-acid residue (or N-degron) of protein substrates. The discovery and subsequent detailed analysis of N-degrons gave rise to the so called N-end rule, which states that the half-life time of a protein is determined by the identity of its N-terminal amino-acid residue. The N-end rule pathway recognizes proteins containing N-terminal destabilizing residues and mediates their polyubiquitination and subsequent degradation in the proteasome. Recent investigations have revealed a role for N-terminal acetylation on the recognition of N-degrons by the N-end rule pathway. Here we summarize these recent findings and highlight the impact on our understanding of the N-end rule pathway with respect to cellular physiology.