"You can’t just proclaim yourself a female and be able to compete against women," she wrote at the time. "There must be some standards, and having a penis and competing as a woman would not fit that standard."
She said her time of reflection has only strengthened her belief that trans athletes have an unfair advantage.
The first transgender woman to win a cycling track world title, Dr Rachel McKinnon, said Navratilova's comments are "deeply ignorant and transphobic".
Over a series of tweets, she responded to Navratilova's remarks, accusing her of "peddling transphobic myths".
Trans advocacy group Trans Actual said it found Navratilova's remarks "devastating", rejecting the claim that trans athletes have an unfair advantage.
In 2016, the International Olympic Committee introduced guidelines allowing trans men to compete without restriction, while trans women must demonstrate that their testosterone level is below a a certain threshold for a least one year before their first competition.
Navratilova was critical of those guidelines, describing them as "unthinkable".
"Simply reducing hormone levels — the prescription most sports have adopted — does not solve the problem," she wrote.
"A man builds up muscle and bone density, as well as a greater number of oxygen-carrying red blood cells, from childhood.
"Training increases the discrepancy. Indeed, if a male were to change gender in such a way as to eliminate any accumulated advantage, he would have to begin hormone treatment before puberty.
"For me, that is unthinkable."
This week, a legal challenge will get underway on proposed rules that would restrict the requirement testosterone levels in female athletes to allow for fairer competition between female athletes.
South African 800m Olympic champion Caster Semenya is challenging the proposed rules by the International Association of Athletics Federation.
The Court of Arbitration for Sport (CAS) in the Swiss city of Lausanne will heard the case from Monday to Friday.
"Ms Semenya is unquestionably a woman. She is a heroine and an inspiration to many around the world," her lawyers said in a statement.