The red palm weevil (RPW), Rhynchophorus ferrugineus (Oliver) is an important pest of palms that causes significant damage by boring into and feeding within palm stem tissues. Here, we studied the proteolytic process of Cry3Aa in the RPW to understand the mechanism of Cry toxicity. The bioassays showed that Cry3Aa toxin is weakly toxic to the RPW. Proteolytic activation assays indicated the Cry3Aa protein is digested into smaller fragments than the 55-kDa activated fragments under different conditions. In particular, at higher mass ratios of gut protease and Cry3Aa protein (5:1, 2:1, and 1:1, respectively), and at 36.9°C for 16 h in a solution of pH 8.6, the Cry3Aa protoxin is over-digested by the gut proteases of weevil larvae. Moreover, the zymogram analysis of the gut proteases revealed the RPW larvae harbors intestinal digestive enzymes mainly composed of serine proteases.This study describes the proteolytic activation process of Cry3Aa in the midgut of RPW larvae.