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Steroidal glycoalkaloids (SGAs) existing in most potato tissues are toxic to humans when the fresh weight is over 200mgkg−1, and high SGAs content would also damage the quality of potato tubers. Sterol side chain reductase 2 (StSSR2) is a key enzyme in the biosynthetic of SGAs in potatoes. To reduce the concentration of SGAs in tetraploid cultivated potato (Solanum tuberosum L.), the gene StSSR2 was edited by the CRISPR/Cas9 system. A total number of 64 mutant lines were generated, and the mutation efficiency was 46%. The percentage of insertion was 6.25%, and the percentage of deletion was 93.75%. Compared to the wild-type (WT), the content of SGAs reduced significantly in the mutated potatoes. The lowest SGAs concentration was 54% of the WT level, which was recorded in the peels of line #360. In the tuber fleshes, the lowest SGAs were 66% of WT level, which was found in line #378. In the leaves, the lowest SGAs concentration was 44% of the WT level as found in lines #204 and #378. In a word, our results showed that the StSSR2 gene of tetraploid cultivated potato could be successfully edited by the CRISPR/Cas9 system to reduce toxic SGAs. [ABSTRACT FROM AUTHOR]