Macropinocytosis is a nonspecific bulk uptake of extracellular fluid. During endosome maturation, the Rab5-to-Rab7 switch machinery executes the conversion from early to late endosomes. However, how the Rab switch works during macropinosome maturation remains unclear. Here, we elucidate the Rab switch machinery in macropinosome maturation using Förster resonance energy transfer imaging. Rab5 is activated and concurrently recruited to macropinosomes during ruffle closure. ALS2 depletion abolishes transient Rab5 activation on macropinosomes, while ALS2 is recruited to macropinosomes simultaneously with Rab5 activation. Thus, we conclude ALS2 activates Rab5 on macropinosomes. The absence of active Rab7 prolongs ALS2 presence and Rab5 activation on macropinosomes, indicating that active Rab7 is necessary for Rab5 inactivation through ALS2 dissociation and plays key roles in the Rab switch on macropinosomes.