Pyruvate dehydrogenase kinases (PDKs) are promising therapeutic targets that have received increasing attentions in cancer metabolism. In this paper, we report the synthesis and biological evaluation of a series of novel dichloroacetophenones as potent PDKs inhibitors. Structure-activity relationship analysis enabled us to identify a potent compound 6u, which inhibited PDKs with an EC value of 0.09 μM, and reduced various cancer cells proliferation with IC values ranging from 1.1 to 3.8 μM, while show weak effect against non-cancerous L02 cell (IC > 10 μM). In the A375 xenograft model, 6u displayed an obvious antitumor activity at a dose of 5 mg/kg, but with no negative effect to the mice weight. Molecular docking suggested that 6u formed direct hydrogen bond interactions with Ser75 and Gln61 in PDK1, and meanwhile the aniline skeleton in 6u was sandwiched by the conserved hydrophobic residues Phe78 and Phe65, which contribute to the biochemical activity improvement. Moreover, 6u induced A375 cell apoptosis and cell arrest in G1 phase, and inhibited cancer cell migration. In addition, 6u altered glucose metabolic pathway in A375 cell by decreasing lactate formation and increasing ROS production and OCR consumption, which could serve as a potential modulator to reprogram the glycolysis pathway in cancer cell.