Here, we determined the sequence of a cDNA encoding a guanylic acid-specific ribonuclease (RNase He1) from Hericium erinaceus that exhibits high sequence identity (59%) with RNase Po1, an enzyme with anti-cancer activity and which is found in Pleurotus ostreatus . RNase He1 and RNase Po1 have similar structures and heat stabilities; hence, RNase He1 may also have potential as an anti-cancer agent. Therefore, we initiated structure-function studies to further characterize the enzyme. Based on the RNase Po1 structure, RNase He1 is predicted to form 3 disulfide bonds involving Cys7–Cys98, Cys5–Cys83, and Cys47–Cys81 linkages. The Cys5Ala mutant exhibited no RNase activity, whereas the Cys81Ala mutant retained RNase activity, but had reduced heat stability. Therefore, the Cys5–Cys83 bond in RNase He1 is essential for the structure of the RNase active site region. Similarly, the Cys47–Cys81 bond helps maintain the conformational stability of the active site region, and may contribute to the greater heat stability of RNase He1.