Speaking on Monday, the Pope warned that blurring distinctions between male and female could lead to the "self-destruction" of the human race.
The comments were "irresponsible and unacceptable", the UK's Lesbian and Gay Christian Movement (LGCM) said.
Vladimir Luxuria, a transgender former Italian MP, called his words "hurtful".
The row erupted as news emerged that the pontiff is to pay his first visit to the Holy Land in May next year.
'Self-destruction'
Pope Benedict made the comments in an end-of-year speech to senior Vatican staff.Defending God's creation was not limited to saving the environment, he said, but also about protecting man from himself.
It was not "out-of-date metaphysics" to "speak of human nature as 'man' or woman'", he said. It came from the "language of creation, despising which would mean self-destruction for humans".
Gender theories, he said, led to man's "auto-emancipation" from creation and Creator.
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