The sugarcane (Saccharum spp. L.) mechanical harvesting system leaves a large amount of straw mulch on the soil surface. The straw mulch may affect soil conditions, root regrowth, and sugarcane yield. Thus, this study assessed the response of sugarcane root system growth and stalk yield to different rates of straw removal. An experiment was conducted in a Rhodic Kandiudox with sand clay loam texture to test the impact of four rates of straw removal: no removal (18.9 Mg ha−1 of dry mass); moderate removal (8.7 Mg ha−1); high removal (4.2 Mg ha−1) and total removal on sugarcane root system and stalk yield. Higher concentrations of roots (60%) were found in the first 40 cm of soil. Moderate straw removal resulted in higher root mass (3.6 Mg ha−1) and stalk production (23 Mg ha−1 of dry mass). However, no straw removal reduced root mass by <40% (2099 kg ha−1) and reduced stalk yield by >20% (105 Mg ha−1). Through regression analysis, it was estimated that retaining between 8.5 and 13 Mg ha−1 of straw resulted in the highest root mass and stalk yield. Managing straw removal to retain a moderate amount enables producers to sustain suitable soil conditions for sugarcane root growth and stalk production while providing straw for industrial use.