Microalgae are promising platforms for sustainable biofuel production owing to their high photosynthetic efficiency and carbon fixation capacity. <i>Nannochloropsis salina</i> is particularly valued for its robust growth and lipid accumulation. However, redirecting carbon flux from carbohydrate to lipid biosynthesis remains a key challenge in microalgal metabolic engineering. In this study, RNA interference (RNAi) was employed to downregulate uridine diphosphate-glucose pyrophosphorylase (UGPase), a central enzyme in chrysolaminarin biosynthesis. After confirming the presence of core RNAi machinery (Argonaute, Dicer, and RDR) in <i>N. salina</i>, an RNAi construct targeting UGPase was introduced. Two transformants, NsRiUGPase 5 and NsRiUGPase 26, were selected through McrBC-PCR and qRT-PCR screening based on reduced methylation-sensitive PCR band intensity and UGPase transcript levels. These RNAi mutants exhibited significantly enhanced growth compared to wild-type. On day 12, dry cell weight (DCW) reached 4.77 g/L in NsRiUGPase 5 and 6.37 g/L in NsRiUGPase 26, representing 32.4% and 76.9% increases, respectively, compared to WT (3.60 g/L). Despite similar lipid contents per biomass, lipid productivity was markedly improved. On day 12, NsRiUGPase 26 achieved 196.3 mg/L/day, a 71.0% increase over WT (114.8 mg/L/day). Fatty acid methyl ester (FAME) analysis showed no significant difference in lipid composition among strains, indicating that UGPase knockdown did not affect lipid quality. These results demonstrate that RNAi-mediated suppression of UGPase successfully redirected carbon flux away from carbohydrate storage toward growth, thereby enhancing overall lipid productivity. This study provides new insights into carbon partitioning in <i>N. salina</i> and underscores RNAi as a powerful tool for microalgal biofuel optimization.