Ofsted chief Sir Michael Wilshaw has warned of gender segregation among teachers in Muslim independent schools.
Inspectors found male and female staff and governors were being divided in Luton's Rabia Boys and Girls School, said the education watchdog.
Last year, Sir Michael wrote to the education secretary with "serious concerns" about staff segregation.
But the Rabia school has strongly rejected the claims and said it was being "unfairly targeted".
Sir Michael has written to Education Secretary Nicky Morgan advising her that for the initial meeting with inspectors "the school insisted on segregating men and women through the use of a dividing screen across the middle of the room".
"This meeting was not carried out in a religious setting but in a classroom."
Sir Michael said inspectors, visiting the independent school earlier this month, "gathered evidence that male and female staff are segregated during whole-school staff training sessions".
"Male staff sit in one room and the session is simultaneously broadcast to female staff in another part of the school," he said.
Inspectors were so concerned, they told the school's proprietor "the school would remain in the inadequate category despite improvements being made elsewhere", said Sir Michael.
Such gender division is against equalities regulations and the requirement for "fundamental British values", he said.
Sir Michael said this requirement is being "flouted" by some independent schools.
"Any form of segregation, without a good educational reason, is likely to lead to an inadequate inspection judgement for leadership and management."
But the Rabia Education Trust has written to the education secretary rejecting Ofsted's claims as "inaccurate" and saying that the school had been "unfairly targeted and harassed".
The letter challenges the account of gender segregation of staff and says that it was unfair to suggest that the school was not meeting the British values requirement.
"We feel extremely disheartened and insecure about the fact that the goalposts are not clear and keep changing," said the trust's letter to the education secretary.
The private school was set up in 1996 to provide an Islamic education for young Muslims in Luton.
The report from inspectors said that such separation of male and female staff does not "demonstrate equality and respect".
But the school, which serves more than 300 pupils aged five to 16, was commended for providing a "good understanding of what it means to be a responsible citizen in modern, democratic Britain".
The inspection report says that pupils have a chance to engage with people from other religions and "pupils demonstrated tolerance of other people's ways of life, even when their own religion views these differently".
A Department for Education spokesman said "It is completely unacceptable for women to be treated less favourably than men, and the advice note we have received from Ofsted on Rabia Girls' and Boys' School is extremely concerning.
"We will consider carefully the inspection report on the school to determine what action to take against any potential breaches in the independent school standards."