With the help of the bacteria in the rumen, ruminants can effectively convert human inedible plant fiber to edible food (meat and milk). However, the understanding of rumen bacteriome in dairy cows is still limited, especially in a large population under the same diet, breed, and milking period. Here we described the sequencing data of 16S rRNA gene of rumen bacteriome from 334 mid-lactation Holstein dairy cows generated using the Illumina HiSeq 2500 (PE250) platform. A total of 24,030,828 raw reads with an average of 71,946 ± 13,450 sequences per sample were obtained. The top ten genera with highest relative abundance accounted for 60.65% of total bacterial sequences. We observed 4,460 overall operational taxonomic units (1,827 ± 94 per sample) based on a 97% nucleotide sequence identity between reads. Totally 6,082 amplicon sequence variants (672 ± 131 per sample) were identified in 334 samples. The shareable datasets can be re-used by researchers to assess other rumen bacterial-related biological functions in dairy cows towards the improvement of animal production and health.