Antimicrobial use in livestock production is linked to the emergence and spread of antimicrobial resistance (AMR), but large-scale studies on AMR changes in livestock isolates remain scarce. Here we applied whole-genome sequence analysis to 982 animal-derived Escherichia coli samples collected in China from the 1970s to 2019, finding that the number of AMR genes (ARGs) per isolate doubled—including those conferring resistance to critically important agents for both veterinary (florfenicol and norfloxacin) and human medicine (colistin, cephalosporins and meropenem). Plasmids of incompatibility groups IncC, IncHI2, IncK, IncI and IncX increased distinctly in the past 50 years, acting as highly effective vehicles for ARG spread. Using antimicrobials of the same class, or even unrelated classes, may co-select for mobile genetic elements carrying multiple co-existing ARGs. Prohibiting or strictly curtailing antimicrobial use in livestock is therefore urgently needed to reduce the growing threat from AMR.