The Pope warned of "powerful political and cultural currents seeking to alter the legal definition of marriage".
He also urged the bishops to emphasise to their Church that premarital s*x and cohabitation was "gravely sinful" and "damaging to the stability of society".
The Pope's comments come shortly after the US states of Washington and Maryland legalised same-s*x marriage.
But at least five US states have scheduled ballot measures that could overturn gay marriage laws.
"S*xual differences cannot be dismissed as irrelevant to the definition of marriage," the Pope said.
He acknowledged during the speech that his point of view may sound "countercultural", especially to young people.
But the Pope stressed that the bishops should do what they could to ensure traditional marriage would be "defended from every possible misrepresentation of their true nature".
In recent weeks, US bishops have also been at the forefront of a campaign against a new rule introduced by the Obama administration that would require health insurance plans to include coverage of contraceptive services.
Timothy Dolan, the New York archbishop who was recently made a cardinal by the Pope, was prominent in that campaign and spoke out during 2011 against the spread of same-s*x marriage laws.
In September 2011, shortly after New York passed legislation, he wrote to President Obama criticising a decision not to support a federal ban on same-s*x marriage.
In the letter, the then-Archbishop Dolan said the White House's policy could "precipitate a national conflict between Church and state of enormous proportions".
