Lysine beta-hydroxybutyrylation (Kbhb) is a post-translational modification induced by the ketogenic diet (KD), a diet showing therapeutic effects on multiple human diseases. Little is known how cellular processes are regulated by Kbhb. Here we show that protein Kbhb is strongly affected by the KD through a multi-omics analysis of mouse livers. Using a small training dataset with known functions, we developed a bioinformatics method for the prediction of functionally important lysine modification sites (pFunK), which revealed functionally relevant Kbhb sites on various proteins, including aldolase B (ALDOB) Lys108. KD consumption or beta-hydroxybutyrate supplementation in hepatocellular carcinoma cells increases ALDOB Lys108bhb and inhibits the enzymatic activity of ALDOB. A Kbhb-mimicking mutation (p.Lys108Gln) attenuates ALDOB activity and its binding to substrate fructose-1,6-bisphosphate, inhibits mammalian target of rapamycin signalling and glycolysis, and markedly suppresses cancer cell proliferation. Our study reveals a critical role of Kbhb in regulating cancer cell metabolism and provides a generally applicable algorithm for predicting functionally important lysine modification sites. Multi-omics analysis of mouse livers shows that protein beta-hydroxybutyrylation (Kbhb) is strongly affected by the ketogenic diet. Combining bioinformatics with experimental validation, a role of Kbhb sites in modulating mammalian target of rapamycin signalling and cancer cell metabolism is identified.