Xanthohumol is a prenylflavonoid from hops with relevant bioactivities. Microbial production has emerged as a sustainable and potentially economic solution to produce it. Herein, we constructed a pathway for the de novo production of xanthohumol in Escherichia coli. Since the xanthohumol pathway depends on the availability of dimethylallyl pyrophosphate (DMAPP) and S-adenosylmethionine (SAM), SAM synthase (metK) was integrated into the genome of E. coli strains with previously engineered DMAPP pathways. Eleven prenyltransferases (PT) and the O-methyltransferase (OMT) from Humulus lupulus (HlOMT1) were tested. E. coli M-PAR-121:BlIDI:metK, constructed by integrating metK into the E. coli strain with integration of isopentenyl diphosphate isomerase (IDI) from Bacillus licheniformis (E. coli M-PAR-121:BlIDI) and expressing CdpC3PT from Neosartorya fischeri and HlOMT1 in combination with the naringenin chalcone pathway, was the best producer. This strain was able to produce 7.3 mg/L of desmethylxanthohumol and 5.3 mg/L of xanthohumol in the bioreactor, representing the first report of de novo production of xanthohumol in E. coli.