AbstractThe effects of hydrophobic chain length on the interfacial and biological properties of diacyl d‐glyceric acid (d‐GA) sodium salts were evaluated based on interfacial tension analyses, dynamic light scattering (DLS), and antitrypsin activity. Of the four synthesized d‐GA‐derived surfactants [dihexanoyl d‐GA sodium salt (diC6GA‐Na), dioctanoyl d‐GA sodium salt (diC8GA‐Na), didecanoyl d‐GA sodium salt (diC10GA‐Na), and dilauroyl d‐GA sodium salt (diC12GA‐Na)], only those with C6, C8, and C10 acyl chains were investigated because diC12GA‐Na were insoluble at room temperature. Together with our previous results, surface tensions at the critical micelle concentration (CMC) were 33.9 mN/m for diC6GA‐Na, 25.5 mN/m for diC8GA‐Na, and 27.9 mN/m for diC10GA‐Na. Evaluation of assembly size via DLS and optical microscopy revealed that diC8GA‐Na and diC10GA‐Na formed large associates with average sizes ranging from 50 to 200 μm at concentrations 4–5 times greater than their CMC, whereas diC6GA‐Na did not form such associates. In tryptic hydrolysis studies using N^α‐benzoyl‐dl‐arginine‐4‐nitroanilide as a substrate, diC8GA‐Na exhibited an inhibitory effect on trypsin (trypsin specific activity: 0.26 ± 0.045 U/mg‐protein) greater than that of diC10GA‐Na (0.39 ± 0.10 U/mg‐protein), whereas diC6GA‐Na did not show antitrypsin activity. These results show that diC8GA‐Na was the most bioactive of the evaluated diacyl d‐glycerate surfactants.