Iron is a metallic element that possesses strong supernatural properties, and is thus used by hunters when combating certain supernatural creatures.
Creatures that Iron affects
- Acheri - Contact with iron will force an Acheri to dissolve and temporarily retreat. 
- Demons- Touching iron will burn a demon, they also can't cross iron, much like salt. Their weakness to iron is so strong, that even Azazel, one of most powerful demons, who was immune to many other demon weaknesses, was still affected by iron (possibly because it was a Devil's trap). However, Alastair wasn't burned by it when he was tied to a devil's trap with it. Astaroth was also able to touch an iron poker with no signs of pain. The only known demon immune to iron is Samhain (it is unknown if iron is effective on Lilith or the Knights of Hell like Abbadon).
- Fairies - All fairies are weakened by iron, although powerful ones like Leprechauns can shrug it off. 
- Ghosts - Shooting or stabbing a ghost with iron will temporarily make the ghost disappear. Ghosts also can't cross iron, much like demons. Sam and Dean considered cold iron to be the best way to repel vengeful spirits. If a person possessed by a spirit touches something made of pure iron the spirit will be forced out of the human.
- Hellhounds- They can be killed using iron, as demonstrated in the episode "Abandon All Hope..." in which Ellen and Jo sacrifice themselves to destroy the attacking Hellhounds by detonating a propane bomb filled with iron nails and rock salt. 
- Leshi - The only way to kill a Leshi, is to cut off its head with an iron axe. 
- Phoenix - Iron burned the Phoenix's skin, and it couldn't touch Iron, or if imprisoned by it, escape. 
- Shtriga - Iron bullets are the only known thing that can kill a Shtriga. 
- Witches - Although Iron doesn't hurt witches, it seems to block their powers, as Dean used it to restrain James, and he was unable to escape from them. 
Iron also affects humans who enter full astralstate, exactly the same as it does to ghosts, they can be imprisoned by it, to touch it causes them pain and makes them temporarily disappear, although like ghosts, they only reappear inside the iron prison.