MSP Amanda Bland has quit Reform UK Scotland to sit as an independent.
The resignation relegates the party to the third-largest group at Holyrood, with one member fewer than Labour.
Bland, who represents Central Scotland and Lothians West as a list MSP, is yet to comment publicly.
Reform UK Scotland have called for the MSP to step down from parliament.
A Reform spokesperson said: "Amanda Bland should reflect on her reasons for choosing to isolate herself from that team of capable individuals.
"It is incumbent on her to resign as a regional MSP for Central Scotland and Lothians West and allow her constituents to be represented by the next Reform candidate on that list."
Under Holyrood's rules, a list MSP would be replaced by the their party's next candidate at the last election if they resigned.
Bland, a former primary school teacher and police officer, was among 17 Reform candidates to be returned to the Scottish Parliament in May as the party made its electoral breakthrough north of the border.
The party finished in joint second with Labour, behind the SNP on 58 seats.
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