Streptococcus thermophilus ASCC 1275, a typical dairy starter bacterium that has been completely sequenced, could produce the highest known amount (~1000 mg/L) of exopolysaccharide (EPS) in milk under the optimal condition among the species of Str. thermophilus. However, little is known about the mechanistic insights into the effects of environmental factors such as pH, temperature and whey peptides on its EPS production. In this study, RNA-seq was applied to this organism cultivated in skim milk under different conditions: 1) Cd1 – pH 6.5 and 37°C; 2) Cd2 – pH 5.5 and 37°C; 3) Cd3 – pH 5.5 and 40°C; 3) Cd4 – pH 5.5 and 37°C with whey peptide supplementation. In condition 1, fermentation was carried out at pH 6.5 at 37°C. Similarly, for condition 2, the fermentation was carried out at pH 5.5 at 37?. For condition 3, the fermentation was carried out at pH 5.5 at 40°C. For condition 4, the fermentation was carried out at pH 5.5 at 37°C with whey peptide supplementation. Samples for RNA seq analysis were collected at 6 h after the start of fermentation. These conditions are based on previous studies carried out in our lab. Total RNA extraction was extracted using Ambion RiboPure-Yeast kit. The high quality RNA from each condition was taken further for transcriptomics library construction. Results showed that 526 genes were upregulated in Cd2 compared with that in Cd1; specifically, ribosomal protein synthesis and EPS assembly genes were upregulated in Cd2 which may explain the enhanced EPS production in Cd2. As for temperature effect, only 49 genes were upregulated in Cd3 compared with that in Cd2; purine and lactose metabolism were enhanced in Cd3 suggesting that more amino sugar may have been synthesized for EPS assembly in Cd3. Sixteen genes were upregulated with whey peptide supplementation in Cd4 compared with that in Cd2. It was found that cysteine and methionine metabolism and some unidentified amino acid/peptide transporters were improved for bacterial growth. This study indicates that regulation of amino acid sugar synthesis, EPS assembly and bacterial growth are responsible for an enhanced EPS production from Str. thermophilus ASCC 1275.