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Before cracking, subjecting water chestnuts to pre-conditioning is a common practice. However, the existing practice of pre-conditioning has no scientific rationale and therefore leads to low flour recovery and poor quality. Thus, the aim of the present research work was to optimize the pre-conditioning process for water chestnuts and to analyze the efficacy of optimized process as a means to increase the resistant starch content and reduce the glycemic index in water chestnut flour. Central composite rotatable design was used to determine the effect of water chestnut:water ratio, water temperature and pre-conditioning time on yield and brightness (L* value) of flour. Optimization of pre-conditioning process was done by response surface of desirability function. Regression models for both the product responses were highly significant (p 0.95). Scanning electron microscopy (SEM) showed compact, smooth and oval shaped starch granules with crystalline network in preconditioned water chestnut flour. FTIR studies also indicated higher absorbance ratio of 1047/1022 cm−1 for preconditioned water chestnut flour which further authenticated the SEM findings. Subjecting water chestnuts to optimized conditions of pre-conditioning (i.e., water chestnut:water ratio 1:1.5; water temperature 87.85̊C and pre-condition time 45 min), also increased the resistant starch content and lowered the glycemic index and glycemic load of the water chestnut flour.