The number of GPs in England fell by 0.6% at the end of last year, with many leaving the profession as the "pressure has become too much", their professional body has said.
The Royal College of GPs said the latest NHS Digital figures show the number of family doctors fell to 33,872 in December from 34,091 in September.
In September 2015 the number of full-time equivalent GPs was 34,592 - a drop of 3%.
RCGP chairwoman Professor Helen Stokes-Lampard said the workload in general practice has increased by at least 16% over the last seven years, but the number of GPs delivering care to patients has not risen in line with this.
She warned that patients suffer from there being fewer family doctors and measures need to be taken to attract new GPs as well as retain existing ones.
"This is very disappointing news - and frustrating as even a small drop in GP numbers can have a huge ripple effect on hard-working GPs, our teams and the care we are able to give to our patients," she added.
"GPs are currently facing intense resource pressures, and we desperately need more doctors if we stand any chance of turning this crisis around.
"For some, the pressure has become too much and it's genuinely awful that some GPs are prematurely leaving a profession which, when properly resourced and funded, can be so rewarding and fulfilling.
"GPs are the cornerstone of our NHS - a system which is the envy of the world - but there is a limit to what we can do and there simply aren't enough of us to deliver the safe care our patients need and deserve."
Shadow health secretary Jonathan Ashworth said the figures were "yet more dismal evidence of the Government letting down patients".
"The fact that GP numbers have fallen again blows apart any claim from Theresa May that the NHS was better prepared than ever before," he added.