Oliver had to cut the lamb's throat before preparing the meat for dinner at an Italian farmhouse.
Before he put the knife in, Oliver said: "I don't know if I can kill a lamb", and then grimaced as he pressed the blade through the animal's windpipe.
Animal welfare group, Advocates for Animals, said Oliver should be "thoroughly ashamed" because the lamb was fully concious when it was slaughtered.
"Slitting a lamb's throat while it is fully concious will undoubtedly cause terrible suffering," a spokesman said.
Not everyone agrees the show should be slammed, though. Animal rights organisation People for the Ethical Treatment of Animals argued that Oliver was right to show the brutality that exists in slaughterhouses.
Sean Gifford, Peta spokesman, said: "In slaughterhouses, the stun gun often misses the mark and animals remain concious. Showing this scene can hit home the message of cruelty."
Channel 4 did warn viewers of its graphic content before the show, which was screened at 8.30pm, before the watershed.
Oliver reasoned later: "A chef who's cooked 2,000 sheep should kill at least one, otherwise you're a fake."
Ofcom said it had received several complaints about the incident and is looking into its Code of Practice to see if the show has broken any guidelines.
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