Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency, and markedly lower kinase promiscuity, than compound C, inhibits cellular AMPK signalling and reduces proliferation of PC3 prostate cancer cells. Biochemical characterization reveals SBI-0206965 is a mixed type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK, and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.