The total number of full-time equivalent GPs in June 2018 was 33,163 - down 533 from 33,686 three months earlier, according to provisional figures from NHS Digital.
The latest drop in the GP workforce takes the government even further from the target set by former health secretary Jeremy Hunt that aimed to boost full-time GP numbers by 5,000 by 2021.
When the target was set in September 2015, the total full-time equivalent GP workforce was 34,592 - more than 1,400 above the level reported for June 2018, and leaving the government 6,500 GPs short of its target.
The latest figures show that between March and June 2018 the GP workforce fell faster than in any other three-month period since the government's workforce target was set.
The figures show that the greatest fall came among partners, with full-time equivalent partner numbers down 315 over the three-month period. A drop of 108 in numbers of GP registrars and smaller declines in numbers of salaried and locum GPs over the period account for the rest of the decrease.
GP leaders have been warning for some time that efforts to boost the general practice workforce are falling short, despite plans to increase recruitment from overseas, boost training places and a drive to boost retention of older GPs.
A survey by the mental health charity Mind published on Thursday found that two in five GPs reported having experienced mental health problems including PTSD in the face of soaring workload and pressure.
RCGP chair Professor Helen Stokes-Lampard said: 'Today’s figures will be demoralising and distressing for GPs and our teams across the country who are striving to deliver care to over 1m patients a day, but without the resources or workforce to do so in a way that is safe for patients, or for themselves.
'Despite great efforts to recruit more doctors to general practice - and we do have more GPs in training than ever before - something clearly is not working, and this must be addressed.'
GPC chair Dr Richard Vautrey said: 'These figures are very concerning as rather than seeing any progress in addressing the GP recruitment crisis as promised by the government, the situation is in fact getting worse.
'The continued decline in the number of GPs across the country is placing extreme pressure on practices and on GPs who are already working above and beyond to meet growing patient demand.'
A DHSC spokesman said: 'We recognise the invaluable contribution of GPs and we are determined to build a strong workforce - that’s why we are investing an extra £2.4bn a year into general practice by 2021, with record number of doctors in training and NHS England planning to recruit an extra 2,000 overseas doctors.'