The commission, the EU's powerful executive arm, said there was nothing untoward about the elevation of Juncker's former chief of staff Martin Selmayr to the post of secretary general, at the head of the EU's 30,000-strong civil service.
The scandal has gained momentum in recent weeks with the European Parliament launching an investigation and warning the affair risks fuelling eurosceptics around the continent.
But the commission insisted Selmayr's appointment was above board and made with the full backing of all EU commissioners.
"The decision was taken by the college of commissioners unanimously, in full compliance with the staff regulations and the rules of procedure of the commission," the commission said in a written response to a list of 134 questions posed by MEPs.
The row centres on what critics say was effectively an instantaneous double promotion for the 47-year-old Selmayr, Juncker's former chief of staff, on February 21.
During a single meeting of commissioners, Selmayr was made first deputy secretary general and then just minutes later secretary general when the incumbent, Alexander Italianer, suddenly announced his retirement.
'Destroys all credibility'
The commission confirmed that Juncker had known of Italianer's plan to retire as early as 2015 and had told Selmayr about it.
But it rejected claims that Juncker and Selmayr had cooked up a plan in November last year to bounce the German into the secretary general role.
It said that technically Selmayr had not been promoted, as he remains on the same civil service grade as before, and that he had taken a pay cut in switching jobs.
As well as the parliamentary probe, the EU ombudsman, which investigates allegations of malpractice in European institutions, has also confirmed it has received two complaints about the matter and is analysing them.
Sophie in 't Veld, a leading liberal member of the European Parliament, said earlier this month the affair "destroys all the credibility of the EU as a champion of integrity and transparency".
The commission, whose 80-page reply to MEPs was put on its website at 3am on Sunday, has denied suggestions the Selmayr process lacked transparency.
And it refused to engage with MEPs' demands about the damage the affair has done to the EU's reputation, saying repeatedly it "does not agree with the premise underlying" the questions.
Juncker insisted on Friday that Selmayr "will not step down", after party sources said he had threatened to quit himself if Selmayr was forced out.
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