The businesswoman has been refused permission for three terraced houses at a former steading next to Kinfauns Castle, east of Perth.
Ms Gloag, one of Scotland's richest women, has been making plans to expand her property empire in the area in recent years.
In 2012, she was given the go-ahead for a six-bedroomed mansion and a three-bedroomed house on the grounds of her estate and last June, she was granted permission for another new-build property near to her home.
However, Perth and Kinross Council has ruled that the location of the latest application at Hill Park in Kinfauns was a green-belt site in an area of great landscape value and could not be built on.
In a written report, planning officers said: "The proposal is within an Area of Great Landscape Value where there is a presumption against built development, except where necessary for operational need.
"No operational need has been demonstrated for this development.
"Development within a green belt area will only be permitted where it can be demonstrated that development is essential for agriculture, horticulture or forestry operations that are appropriate to the green belt.
"No justification in this regard has been submitted.
"The economic impact of the proposal is not likely to be significant and will largely be confined to the construction phase of the development."
Ms Gloag, 71, wanted to erect a row of three-bedroomed one-and-a-half storey houses at the site.
She was originally granted permission for the development in 2008 but building work never started and she had to apply again when her consent expired.
The application had previously attracted objections from the Architectural Heritage Society of Scotland.
In a letter to planners, the conservation body said: "This proposal breaches just about every planning policy for the location.
"There is no apparent operational need and it would impair the adjacent landscape. The society wishes to object to the application in the strongest possible terms."
The two homes Ms Gloag applied to build in 2012 also attracted criticism.
Forestry Commission Scotland warned the buildings would ruin an area of ancient woodland and neighbours objected to the proposal, claiming it would spoil the privacy and seclusion they currently enjoy and cause a drop in property values in the area.
Ms Gloag, who has an estimated £650 million fortune with her brother Sir Brian Souter, won the right to restrict access to her property in 2007.
The case, the first of its kind, allows her to keep the public out of 10 acres of grounds.
Although she remains a non-executive director of the transport empire she built with her brother, Ms Gloag now devotes much of her business acumen towards humanitarian causes.
In the 2004 New Year's Honours list she was awarded an OBE for her charity work and in 2011 she became the first Scot to be honoured with a prestigious international award.
The former nurse received the Eleanor Roosevelt Val-Kill Medal, whose previous honorees include US Secretary of State Hillary Clinton and Queen Noor of Jordan.
A spokeswoman for Ms Gloag said she did not wish to comment.