Natural sweeteners, such as agave syrup, might be a healthy alternative to sucrose used in sweet bakery products linked to obesity. We evaluated the effect of sucrose replacement by agave syrup on rheological and microstructural properties of muffin batter and on physical and sensorial properties of the baked product. Muffins were formulated by replacing 25%, 50%, 75%, and 100% of sucrose by agave syrup (AS) and partially hydrolyzed agave syrup (PHAS), and by adding xanthan gum and doubled quantities of leavening agents. Rheological and microstructural properties of batter during baking were analyzed over the range of 25–100 °C. In the muffins, the structure, texture, color, and sensory acceptance were studied. The combination of agave syrup with xanthan gum and doubled quantities of leavening agents affected (p < 0.05) rheological and microstructural properties of the batters and textural properties of the low-sucrose muffins compared to the controls. The increase in agave syrup levels resulted in a darker crumb and crust. Sensory evaluation showed that AS-75 and PHAS-75 were the best alternatives to the control samples. Our results suggest a plausible substitution of up to 75% of sucrose by agave syrup in preparation of muffins, with physical and sensorial characteristics similar to those of their sucrose-containing counterparts.