Microtubers are valuable tools to be used in the researches of potato. Among the factors sucrose is the most critical stimulus for potato microtuber production in vitro. The aim of our study was to define whether only sucrose was enough to induce the tuber and how it impacted on physiological responses in potato in vitro. Among sucrose concentrations chosen 80 and 100g/l sucrose could induce maximum tubers in three cultivars of Solanum tuberosum L., Atlantic, Desiree and 851 in vitro at 22 oC under 16-h photoperiod. Meanwhile tuber induction percentage showed no difference in MS with 80 and 100g/l sucrose among 0, 16 and 24-h photoperiod and cuttings with no nodes induced no tubers in cv. Atlantic. Therefore, 80 or 100g/l of exogenous sucrose concentrations and cuttings with node were essential to in vitro tuber induction in potato. The endogenous sucrose content and acid invertase in induced cuttings were higher than corresponding tubers and dramatically increased with the increase of sucrose concentration in MS. Starch content was completely opposite to endogenous sucrose content and decreased with the increase of sucrose concentration in MS. Sucrose synthase (Susy) and sucrose phosphate synthase (SPS) accumulated only in tubers, but no significant differences of Susy and SPS were between 80g/l and 100g/l sucrose. In conclusion, high levels of exogenous sucrose resulted in a dramatic high gradient of endogenous sucrose content between inducing cuttings and the corresponding tubers, which was beneficial to trigger differential activity expression of sugar metabolizing enzymes and to accumulate starch in tubers in vitro.