Cyanogenic glycosides are a large group of secondary metabolites that are widely distributed in the many plants commonly consumed by humans, birds, and other animals. Thiosulfate sulfurtransferase (TST) and 3-mercaptopyruvate sulfurtransferase (MPST), are two evolutionary-related enzymes that constitute the defense against cyanide toxication and participate in the production of sulfane sulfur-containing compounds. The expression and activity of TST and MPST as well as the level of sulfane sulfur in chicken tissue homogenates of the liver, heart, and gizzard were investigated. The highest expression/activity of TST and MPST was noticed in liver homogenates which was associated with the high sulfane sulfur level. Both the expression and activity of TST as well as the sulfane sulfur level in chicken gizzard homogenates were significantly lower than in the liver and heart. Both TST and MPST enzymes can play an important role in cyanide detoxification in chicken tissues. Maintaining appropriate sulfane sulfur level together with the high activity of these enzymes is essential to protect tissues from the toxic effects of cyanide, released from certain nutrients.