The demand for rice varieties with lower amylose content (AC) is increasing in Southeast Asia, primarily due to their desirable texture and cooking qualities. This study presents the development of whole-grain rice lines with low to intermediate glycemic index (GI) and reduced AC. We selected six rice lines for in vivo GI assessment based on their starch properties. We successfully identified two lines with low AC that exhibited low and intermediate GI values, respectively. Our findings indicate that dietary fiber (DF) content may significantly influence rice GI. The selected whole-grain low-GI line showed a higher ratio of soluble dietary fiber (SDF) to insoluble dietary fiber (IDF) compared to control varieties, highlighting SDF’s potential positive role in lowering whole-grain rice’s GI. This study underscores the feasibility of developing rice varieties with desirable agronomic traits, nutritional traits, and culinary attributes, particularly for individuals managing their blood sugar levels. Additionally, we proposed the positive role of starch composition, especially DF content, in modulating the GI of rice. This study reinforces the importance of incorporating starch properties and DF content into rice breeding programs to produce more health-oriented and marketable rice varieties.