Selenite (H2SeO3) reacts with thiol compounds (RSH) under acidic conditions to form selenotrisulfides (RSSeSR, i.e. monoselenodithiols). The stoichiometry of the reaction is proposed as 4RSH+H2SeO3→RSSeSR+RSSR+3H2O. Surprisingly, we found novel polynuclear selenium-containing compounds, i.e. polyselenodipenicillamines (PenSSe2–4SPen), in the reaction of D-penicillamine (PenSH) with H2SeO3. The selenium-centered features of PenSSe2–4SPen were determined by 1H-NMR and LC-MS/MS analyses, showing that the selenium isotope abundance patterns of the compounds were in good agreement with the theoretically-calculated ones. In order to better understand the mechanisms for PenSSe2–4SPen production, various molar ratio of H2SeO3 (1/8 to 4 times of PenSH) was reacted with PenSH, and the concentration of the products was calculated from integral values of dimethyl proton signals for PenSSe1–2SPen as compared with methyl proton signals for acetic acid (an internal standard). Total PenSSe1–2SPen concentration was increased with increasing of H2SeO3, in which concomitant decrease of PenSSPen (disulfide form of PenSH) was observed. Based on these results, we proposed the PenSSe2–4SPen production mechanisms being involved in penicillamine selenopersulfides (PenSSe1–2H).