Reading's departure - to become a non-aligned institution outside any mission group - leaves the 1994 Group with just 11 active members.
In August, four universities left the 1994 Group for the Russell Group: Durham, Exeter, York and Queen Mary, University of London. And in the autumn, the University of Bath, University of St Andrews and the University of Surrey departed to become non-aligned.
Reading's membership formally expires in July 2013 but it will cease to play an active role in the group from today.
Sir David Bell, Reading vice-chancellor, said: "We have been proud to be a member of the 1994 Group since its inception 18 years ago. As an organisation for research-intensive universities with a strong focus on the student experience, the 1994 Group has done an effective job in influencing government policy and promoting best practice.
"However, in these uncertain times in higher education, we do not wish to be tied into any particular group. Rather, we would prefer to engage in partnerships with a much broader group of universities."
This year has seen disagreements between 1994 Group members as to whether the group should take up near-identical policy positions to the Russell Group, or try to forge a more distinctive identity based around the student experience offered by its smaller-scale institutions.
Reading's departure comes less than a year after Sir David, previously permanent secretary in the Department for Education, took charge of the institution.
"As a leading university, we are confident about our place both nationally and internationally," he said. "Here in the UK, we will remain a strong voice for diversity and breadth in the research-intensive part of the higher education sector and continue to champion an intellectually challenging and demanding experience for all students."
The 1994 Group has also seen several staff departures this year, with chief executive Paul Marshall and director of research Rachel Winzer leaving, and director of communications Mark Fuller about to leave.