Exo-polysaccharide (EPS) produced by dairy starters plays critical roles in improving texture and functionalities of fermented dairy products. One of such high EPS producers, Streptococcus thermophilus ASCC 1275 (ST1275) was used as a model dairy strain to understand the stimulation of its EPS production under optimal milk fermentation conditions. The mRNA-seq analysis and targeted pathway analysis indicate that genes associated with lactose (milk sugar) catabolism, EPS assembly, proteolytic activity, and arginine/methionine/cysteine synthesis and transport in ST1275 were significantly up-regulated under the optimized conditions of pH 5.5, 40°C, or WPI supplementation compared to that of pH 6.5 and 37°C, respectively. This indicates that genes involved in above metabolisms cooperate together for improving EPS yield from ST1275. This study provides a global view map on potential targeted pathways and specific genes accounted for enhanced EPS production in Str. thermophilus and that could be modulated by fermentation conditions.