The Hollywood star, 62, stood in the Court One dock of the Old Bailey to deny five allegations relating to three men, who are now in their 30s and 40s.
Spacey was listed under his full name of Kevin Spacey Fowler and appeared in person having been granted unconditional bail.
Wearing a blue suit and blue tie with pink polka dots, he spoke to confirm his name and date of birth.
He then spoke clearly as he denied four charges of sexual assault and one count of causing a person to engage in penetrative sexual activity without consent.
The alleged offences are said to have taken place in London and Gloucestershire between 2005 and 2013.
At the time, Spacey was artistic director of The Old Vic theatre in London.
The House of Cards star has an address in Waterloo, south London, and in the US, where he has family and a nine-year-old dog.
Last month, at an initial court hearing at Westminster Magistrates' Court, Spacey's lawyer Patrick Gibbs QC said his client "strenuously denies any and all criminality in this case".
Gibbs said Spacey returned to the UK "to establish his innocence" and "proceed with his life".
The charges include two counts of sexual assault on a man, who is now in his 40s, in March 2005 in London; sexual assault on a man, who is now in his 30s, and causing him to engage in penetrative sexual activity without consent, in August 2008 in London; and sexual assault on a man, who is now in his 30s, in April 2013 in Gloucestershire.
The Crown Prosecution Service authorised the charges against Spacey in May, but he could only be formally charged after travelling to the UK last month.
Spacey, a two-time Academy Award winner, is known for starring in American Beauty and The Usual Suspects.
He was artistic director at The Old Vic between 2004 and 2015.
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