To decrease the accumulation of reducing and non-reducing sugars in potato tubers stored at low temperature, a multiple gene-silencing vector pBIPhLSAR1-IR possessing a part of starch phosphorylase L and starch-associated R1 genes was constructed and transformed into potato (Solanum tuberosum L.) cultivars Agria and Marfona. Polymerase chain reaction (PCR) and southern blotting indicated that the RNAi construct was transformed successfully into the genome. Real-time reverse transcription PCR (RT-PCR) analysis showed that the expression level of starch phosphorylase L and starch-associated R1 genes was reduced in transgenic microtubers in relation to the non-transgenic plants. The accumulation of total and reducing sugars in transgenic microtubers was significantly decreased (up to 58% in line AM6) compared to the control. Repression of these genes decreased the phosphate content of starch and increased starch content in transgenic microtubers, implying that silencing of starch phosphorylase L and starch-associated R1 genes reduced starch breakdown during cold storage conditions.