The appointment, announced on Thursday, places Muyingo at the helm of one of government's largest and most consequential ministries as the First Lady continues her recovery.
"In exercise of the powers vested in the President under Article 99(1) of the Constitution of the Republic of Uganda, H.E. Yoweri Kaguta Museveni has appointed Hon. John Chrysostom Muyingo as the Acting minister of Education and Sports in the absence of a substantive Minister," the statement by the Presidency reads.
It added that the appointment takes immediate effect.
Mrs Museveni was reappointed Minister of Education and Sports during last month's Cabinet reshuffle but has not resumed active duties. She did not appear before Parliament's Appointments Committee for vetting and was also absent during the swearing-in of ministers, fueling speculation about her health.
The development came a day after President Museveni publicly revealed that the First Lady had fallen critically ill in March, marking the first official acknowledgment of her prolonged absence from public life.
In a heartfelt message celebrating Mrs Museveni's 78th birthday on Wednesday, the President disclosed that she had battled a serious illness but had since overcome it.
While he did not reveal the nature of the ailment, Museveni described the period as one of great personal and family concern, praising the First Lady's resilience and faith during her recovery.
"She became critically ill in March but, by the grace of God, overcame it," the President said in his birthday tribute.
The revelation helped explain months of absence by the First Lady, who has not been seen at major government functions since March.
Muyingo's appointment is expected to ensure continuity at the Education Ministry as government prepares for key policy decisions affecting schools, universities, sports administration and ongoing education sector reforms.
A longtime education administrator and legislator, Muyingo has served as State Minister for Higher Education and is widely regarded as one of the ministry's most experienced officials, having overseen several higher education policy initiatives over the years.
His elevation comes amid a broader effort by President Museveni to fill leadership gaps in ministries affected by delays in the approval of substantive ministers.
On Wednesday, the President also appointed State Minister for Microfinance Haruna Kasolo Kyeyune to act as Minister of Foreign Affairs.
The Foreign Affairs docket has remained without a substantive political head after Parliament declined to approve the appointments of Adonia Ayebare as Minister of Foreign Affairs and Calvin Echodu as State Minister for Foreign Affairs during the recent vetting process.
The temporary appointments signal a move by State House to ensure uninterrupted operations in critical ministries as questions surrounding the composition of the new Cabinet continue to be addressed.
For the education sector, however, attention is likely to remain focused on the health and eventual return of Janet Museveni, who has served as Minister of Education and Sports since 2016 and remains one of the most influential figures in President Museveni's administration.