Streptococcus thermophilus is a common starter in yogurt production and plays an important role in the dairy industry. In this study, a galactose-positive (Gal + ) mutant strain, IMAU20246Y, was produced using the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine (NTG) from wild-type S. thermophilus IMAU20246, which is known to have good fermentation characteristics. The sugar content of milk fermented by either the mutant or the wild type was determined using HPLC; metabolism of lactose and galactose was significantly increased in the mutant strain. In addition, we used response surface methodology to optimize components of the basic M17 medium for survival ratio of the mutant strain. Under these optimal conditions, the viable counts of mutant S. thermophilus IMAU20246Y reached 4.15 × 10 8 cfu/mL and, following freeze-drying in the medium, retained cell viability of up to 67.42%. These results are conducive to production of a high-vitality starter culture and development of "low sugar, high sweetness" dairy products.