Starch initiation has been intensively investigated. In tubers of Solanum tuberosum, it is controlled by a heteromultimeric isoamylase complex composed of StISA1 and StISA2. Moreover, two non-catalytic starch binding proteins (i.e, ESV1 and LESV) regulate leaf starch metabolism in Arabidopsis. Both proteins interact with starch glucans through a C-terminal antiparallel β-sheet domain and differ by the presence of a 115 amino acid N-terminal overhang in LESV. On the other hand, LESV was shown to target ISA1 to storage starch in Oryza sativa seeds through protein-protein interaction. Here were report on the CRIPSR/Cas9 inactivation of StLESV or StESV1 in potato and the characterization of the respective tuber starches. Interestingly, starch from esv1 mutants was unaffected whereas granule diameter was severely reduced in lesv plants. This was negatively correlated to the number of granules, coherent with a starch initiation phenotype. Strikingly, scanning electron microscopy of purified starches revealed that lesv plants phenocopy Stisa1 and Stisa2 antisense lines described 20 years ago by Bustos and collaborators. These data show that LESV is necessary for regulating starch initiation in potato tubers likely by interacting both with the StISA1/StISA2 complex and newly initiated glucans. The lack of the N-terminal tail in ESV1, together with the absence of phenotype in esv1 mutants, lend support on the role of the LESV N-terminal overhang in interacting with ISA1.