We investigated the molecular weight (MW) range that stimulates gastric secretion by fractionating skipjack broth (SJT-Br), a gastric secretion stimulant, by MW. Pepsin-like protease (PLP) activity and secretion induced by fractions of over 14,000 Da, 6,000-14,000 Da, 3,500-6,000 Da, 2,000-3,500 Da, 1,000-2,000 Da, and 100-1,000 Da were determined by infusions into the ex vivo stomachs of black rockfish, and compared with artificial seawater (ASW, control) and SJT-Br. The results showed that the fraction over 14,000 Da did not stimulate the ex vivo stomach of black rockfish, and the activity and secretion of PLP were significantly lower than SJT-Br and comparable to ASW. In contrast, rapid stimulation of gastric secretion was observed with fractions of 2,000-3,500 Da and 3,500-6,000 Da. Thus, SJT-Br appears to contain bioactive peptides, which is consonant with numerous reports finding that fish protein hydrolysate (FPH) has bioactive properties. Future studies should further examine the gastric stimulant effects of these MW fractions in live fish, in order to evaluate their growth-promoting effects in vivo. Boiling broth and FPH discarded during fish processing may be valuable sources of bioactive molecules that could contribute to developing highly efficient aquaculture fish feeds.