In the present study, the effects of exogenous ascorbic acid (AsA) treatments on potato microtuber formation and the expression of tuberization-related genes, as well as the influence of knockout of the key tuberization-related gene Solanum tuberosum self-prunning 6A(StSP6A) on the AsA-induced tuberization were investigated. Two diploid landraces CIP-149 (Solanum phureja), CIP-178 (S. ajanhuiri) and tetraploid C-88(S. tuberosum) were treated with different concentrations (0，1，5，10，20，50 mmol/L) of AsA. The results showed that exogenous AsA treatments with 1 mmol/L to 5 mmol/L could significantly induce tuber formation. The expression analysis of 10 tuberization-related genes demonstrated that the exogenous 1 mmol/L AsA treatment also significantly affected the expression of those tuberization-related genes, in which a general trend was demonstrated with an enhancement of positive regulator or repression of negative regulator expression for tuber formation as compared with the control, especially StSP6A, which was extremely significantly up-regulated during the early stage of tuber formation and AsA treatment. Knockout of StSP6A resulted in elimination of the effect of exogenous AsA-induced tuberizations in potato. These findings indicated that AsA-induced tuber formation is triggered by regulating the tuberization-related gene expression, and StSP6A played key roles in AsA-induced tuber formation in potato.