The killing reduced public support for the Vietnam war in the US and became a turning point in the conflict.
All along Calley has refrained from commenting about the massacre in public but broke his silence on Wednesday after accepting an invitation to speak at a local community club.
Calley spoke on Wednesday, but due to a restricted audience his remarks did not become known until Saturday.
"There is not a day that goes by that I do not feel remorse for what happened that day in My Lai," he told members of the Kiwanis Club of Greater Columbus, Georgia.
Calley was sentenced to life in prison but his sentence was later reduced to house arrest.
He was freed after three years when Richard Nixon, the then US President intervened.
When asked on Wednesday if obeying an unlawful order was not in itself an unlawful act, he said: "I believe that is true."
"If you are asking why I did not stand up to them when I was given the orders, I will have to say that I was a second lieutenant getting orders from my commander and I followed them - foolishly, I guess."
Calley's immediate commander at that time was Captain Ernest Medina, who was also tried in connection with the massacre.
But Medina, represented by famous defense attorney F. Lee Bailey, was acquitted of all charges in 1971.
A survivor of mass killings by US troops in the Vietnamese village of My Lai in 1968 has said he welcomed the public apology made by a former officer convicted for his role in the atrocity.
"It's a question of the past and we accept his apologies, although they come too late," Pham Thanh Cong, who saw his mother and brothers killed in the massacre, told the news agency AFP on Saturday.
"However, I prefer that he send his apologies to me in writing or by email."
Pham Thanh Cong, who is the director of a small museum at My Lai, said: "I want him to come back... and see things here.
"Maybe he has now repented for his crimes and his mistakes committed more than 40 years ago."