Hyaluronic acid (HA) is a highly polymerized linear polysaccharide widely employed in cosmetics, pharmaceuticals and food. Streptococcus zooepidemicus, the primary HA industrial strain, faces high production costs and difficulties in regulating molecular weight (MW). To enhance HA yield, lactate dehydrogenase gene (ldh) was knocked out. Surprisingly, the knockout strain demonstrated a significant reduction in HA MW. To investigate the underlying mechanisms and identify novel factors affecting HA MW, transcriptome sequencing was performed on both the wild-type and knockout strain. The results revealed altered transcript levels for three presumptive cysteine transporter genes, fliY1/2/3. To assess their roles in HA synthesis, fliY1/2/3 were individually knocked out or over-expressed. It was found that overexpression of fliY1 reduced the MW further to 0.8 × 10