May and finance minister Philip Hammond met around 150 executives to discuss Britain's departure from the world's biggest trading bloc and this week's annual budget, which included spending pledges and higher taxes on technology firms.
Serco CEO Rupert Soames said both May and Hammond had been "reassuringly robust" and pragmatic in the meeting where May sought to assuage the concerns of business over the imminent departure from the EU.
"(It was) helpful to have practical recognition that the uncertainty is causing investment to slow down," he told Reuters.
With five months to secure a deal before Britain is due to leave the EU, business leaders are demanding certainty over the kind of trade terms the divorce will deliver. In particular, they want reassurances that there will be a 21-month transition period after Britain is due to leave the bloc.
Attendeees at the meeting at Guildhall in London said the atmosphere was good.
"There was a sense that they've got their mojo back," Legal & General chief executive Nigel Wilson told BBC News after the meeting.
"There was assured confidence from both of them that Brexit negotiations will go well, so we'll get a good solution, that it will go through parliament in a reasonably smooth way."
If Britain and the EU fail to strike a withdrawal agreement, there will be no formal transition period to cushion the impact of Brexit.
Earlier on Wednesday, the opposition Labour Party's finance spokesman John McDonnell also briefed businesses on the budget, and said those attending had quickly focused on Brexit during the discussion.
"We're as worried as they are about the uncertainty and the lack of assurance coming from government about a deal," McDonnell told reporters after the meeting.
The Brexit negotiations are currently stalled over a disagreement on a fallback plan to avoid a hard border on the island of Ireland, former focal point for sectarian tensions, if a future trading relationship is not in place in time.
The latest meeting with business leaders follows a conference call the prime minister held with a similar-sized group of business leaders earlier this month.
May is bidding to to prove her business-friendly credentials. She told the Conservative party conference that she would "back business" afer former foreign minister and prominent Brexiteer Boris Johnson reportedly used a different four-letter word to express his disdain for businesses' interventions on Brexit.
"She set out that the government would work to build a Britain that would be unequivocally pro-business," May's office said in a statement after the meeting. (Reporting By Andrew MacAskill, William James, Alistair Smout and Elisabeth O'Leary; Editing by Guy Faulconbridge, Richard Balmforth)
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