WorldPride, a week-long international festival with a parade, conference and exhibitions, is due to start on August 6.
Hagai El-Ad, of Jerusalem Open House, which is organising the event, said on Tuesday: "Jerusalem's holiness shines at its best when all human beings are respected equally in the city.
"We are determined and committed to hold the WorldPride event in Jerusalem as planned."
Homosexuality is offensive to many Muslims, Jews and Christians, all of whom have religious sites in Jerusalem.
Some opponents of WorldPride put up posters offering a $4,500 reward for anyone who kills "one of the people of Sodom and Gomorrah".
An ultra-Orthodox Jew stabbed and wounded three participants at the event last year.
The focus of opposition is the planned parade, though organisers dismiss local rumours that marchers would try to reach Jerusalem's holiest site, known to Jews as the Temple Mount and to Muslims as al-Haram al-Sharif (Noble Sanctuary).
Adnan Husseini, director of the Muslim Waqf, which administers the sanctuary, said: "We are living in tough days, such events are not ones Jerusalem needs."
The Temple Mount is the holiest site in Judaism and is also important for Christians. Israel's Sephardic chief rabbi, Shlomo Amar, has appealed to Pope Benedict to speak out against the parade. The Vatican has already expressed its opposition.
Freedom of speech
Evangelical Christian groups call the festival a "calculated and confrontational act meant to provoke and offend".
City authorities have left the decision on whether the parade can go ahead to the police.
El-Ad said: "It is really a question of freedom of speech. If the police cannot protect peaceful marchers in a major street in Jerusalem then who is in control of the city?"