In headcount terms, the number of GPs in England fell from 41,865 in September 2016 to 41,475 in December, a drop of 390 - or 0.9%.
Full-time equivalent (FTE) data show that the GP workforce fell 445 from 34,495 to 34,050 over the three months to 31 December 2016 - a 1.3% drop.
Excluding registrars and locums there were 28,204 FTE GPs at 31 December 2016, down from 28,530 in September.
The findings published by NHS Digital are the latest blow to the government's ambition of increasing the GP workforce by 5,000 by 2020/21, and come just a day after the independent doctors and dentists review body (DDRB) warned that it is 'unclear' how GP services can be maintained over the coming years given the growing GP workforce crisis.
Reaction: Workforce crisis deepening, say GPs
The figures suggest a growing trend of falling FTE GP numbers, coming after a drop of just under 100 FTE GPs over the 12 months to September 2016.
Analysis by GPonline found that GP numbers fell fastest in the South London NHS region, where the number of full-time equivalent GPs dropped 77 to 2,190 between September and December 2016 - a 3.4% decline.
In the North East region of England, FTE GP numbers dropped 3.1% - down by 47 to 1,464 over the three-month period.
Of 13 NHS regions across England, just two - Wessex and the West Midlands - avoided a drop in the FTE GP workforce, with an increase of five FTE GPs and six FTE GPs respectively.
In Kent, FTE GP numbers slipped by 0.9%, while in every other area numbers fell by more than 1%.
A total of 18% of headcount GPs are aged over 55, the workforce data reveal. However, the age of 7% of GPs is unknown, and if this group is excluded, 19% of the workforce whose age is known is 55 or over.
One positive statistic is that 48% of GPs overall are aged under 45. A total of 15% of the headcount workforce are aged 30-34, 17% 35-39 and 16% are aged 40-44.
Partners now make up just 56.9% of the total headcount number of GPs, the figures reveal, with 26.6% of GPs listed as 'salaried/other' GPs, while 13% are registrars. The rest are locums (3.8%) or retainers (0.4%).
The figures suggest that men and women make up almost equal proportions of the FTE workforce, with 47.2% of FTE GPs male, compared with 46.6% of FTE GPs female.
A DH spokeswoman said: 'GPs are the absolute bedrock of the health service and we remain committed to an extra 5,000 doctors in general practice by 2020.
'We have made important strides over the last year to improve conditions to attract more GPs - such as paying a large amount of GP indemnity costs, cutting red tape, agreeing a new contract with their union to deliver a 1% pay rise, as well as bringing in new schemes to help GPs work more flexibly towards retirement.
'We currently have the highest number of GPs in training than ever before, and we know that it will take time for this to impact on GP workforce numbers. We will also take strong action to make sure the almost 600 practices who have not yet reported their workforce numbers do so.'