Abstractγ-aminobutyric-acid (GABA) is a mental health-supporting substance that helps release anxiety and depression and improves memory. Lactiplantibacillus plantarum SKKL1, a GABA-producing bacterium, has been introduced to formulate a gut-brain axis product. However, growth and sugar consumption of L. plantarum SKKL1 in milk were ineffective. This obstacle was investigated by varying different types of milk, sugars, fermentation temperatures, and times. The results revealed that none of these parameters improved growth and bacterial metabolism in milk, except addition of soluble protein as found in yeast extract and malt extract. Although a protease deficiency of L. plantarum SKKL1 was discovered, it was not a primary barrier to cell propagation. Insight of this study showed clearly that soluble protein was an essential metabolic activator for growth, nutrient consumption, and protease synthesis, then stimulated lactic acid and GABA productions. While, milk casein and casein hydrolysate, a complex protein structure with low solubility, were not utilized by L. plantarum SKKL1. The novelty of this study is the first in-depth investigation to confirm a significant effect of soluble protein on enrich-GABA milk fermentation by L. plantarum SKKL1 as the sole starter without protease and monosodium glutamate addition.