Growth-promoting antibiotics have been used in cattle, but concern about antimicrobial overuse has prompted a re-evaluation of this practice. To evaluate changes in the ruminal microbiota of feedlot cattle by virginiamycin, a total of 76 crossbreed beef cattle from 2 batches of cattle at different sampling periods (B1 and B2) were divided into 2 groups: one receiving virginiamycin in their diet (ATB) and the other receiving the same diet without any growth promoter (CON). The use of virginiamycin was associated with significant changes in the diversity and composition of the ruminal microbiota of cattle in B1, but not in cattle in B2. Several bacterial taxa were significantly more abundant in samples from CON, ., an unclassified genus of the TM7 phylum, whereas others were associated with the use of virginiamycin, and spp. In conclusion, virginiamycin can affect bacterial diversity and composition in the rumen of feedlot cattle, but its effect is inconsistent in different seasons of the year.