Abstract: Gayam seed (Inocarfus fagifer Forst.) has a high amylose content and is a good source of resistant starch (RS). Pre-gelatinization during the preparation of gayam flour can increase the RS content, which is associated with a lower glycemic index (GI). The objective of this research was to study the changes in both the RS content and GI of gayam flour with pre-gelatinization treatment. Pre-gelatinized gayam flour was prepared from unpeeled gayam seed that was boiled at 100°C for 15, 30, or 45 min. The results showed that the RS contents of pre-gelatinized gayam flour (by dry weight analysis) boiled for 0, 15, 30 and 45 min were 15.78±0.99, 16.95±0.54, 17.06±1.10 and 17.92±0.39%, respectively. The RS content of pre-gelatinized gayam flour boiled for 15 and 30 min did not differ significantly compared with the control, whereas pre-gelatinization for 45 min produced the highest RS content. Using glucose as the reference food, the GI values of pre-gelatinized gayam flour boiled for 0, 15, 30 and 45 min were 74, 75, 61 and 57, respectively. Thus, a longer pre-gelatinization time can produce a lower GI value for gayam flour, which is associated with an increased RS content.