In a video posted to the embassy's Facebook page and YouTube channel on Thursday, James "Wally" Brewster and his spouse, Bob Satawake, called for an end to discrimination.
It marked the first time the ambassador has publicly addressed the controversy that erupted after President Barack Obama named him to the post last year.
Roman Catholic and evangelical leaders have criticized the appointment as being out of line with the majority-Catholic Dominican Republic's values.
The controversy peaked last summer when the country's only Catholic cardinal, Archbishop of Santo Domingo Nicolas de Jesus Lopez Rodriguez, referred to Brewster using an anti-gay epithet in remarks to the press.
"Has it always been easy? No," Brewster said in the video commemorating LGBT Pride month, held each June. "Of course, there were those that were not so kind prior to our arrival and after we landed," he said.
"To those individuals who continue to discriminate against individuals because of who they are as human beings, I have to ask, 'Isn't it time to stop hating?'" he added.
Brewster, who arrived six months ago to take up his post, is one of several openly gay U.S. ambassadors appointed by Obama.