Rabia Girls' and Boys' school in Luton, Bedfordshire, does not reflect the "spiritual, moral, social and cultural standard" expected, the schools watchdog Ofsted said.
In a letter to Education Secretary Nicky Morgan, Ofsted chief Sir Michael Wilshaw said inspectors themselves were segregated by a dividing screen during a recent meeting at the school.
Addressing Morgan as both Education Secretary and Minister for Women and Equality, Sir Michael wrote: "[Her Majesty's Inspectorate] who inspected the Rabia Girls’ and Boys’ School in Luton expressed their concern when, at 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. HMI also gathered evidence that male and female staff are segregated during whole-school staff training sessions.
..the school insisted on segregating men and women through the use of a dividing screen across the middle of the room Ofsted
"Male staff sit in one room and the session is simultaneously broadcast to female staff in another part of the school.
"HMI were so concerned about the behaviours modelled by the leaders of this school they informed the proprietor that the school would remain in the inadequate category despite improvements being made elsewhere.
"This sort of behaviour manifested by the leaders of this school clearly does not conform to the spirit of the equalities legislation which underpins the spiritual, moral, social and cultural standard."
The school prompted controversy last year when an Ofsted inspection found girls and boys were not treated equally.
In one example, design and technology teaching at the school limited girls to "knitting and sewing".
The school currently teaches more than 300 five to 16 year olds.
It was founded in 1996 as a private school.
A DfE spokesman: "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 have referred this case to the EHRC so they can consider whether the school has breached the Equalities Act, and 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."
In January Ofsted said that, despite concerns over segregation, the school "is beginning to address weaknesses found at the previous inspection with regard to teaching pupils about what it means to be a good citizen in Britain."
Rabia School responded to refute some of the allegations, saying: "We and our pupils are left having to pick up the pieces of this substandard inspection and letter".