"Put your ambitions aside and act in the interests of the country and the constitution by publicly stepping down," she said in an open letter to Zuma on Thursday.
The letter comes a day before Zuma's legal team makes representations to the National Prosecuting Authority.
Zuma faces charges of fraud, corruption, money laundering and racketeering related to a multi billion rand government arms deal.
Zille said that should Zuma be elected president without first having been exonerated — by a court — of the charges against him, the constitution would be seriously undermined.
"In fact, I have been reliably informed by senior members of the Bar that your election could be challenged in the Constitutional Court.
"That is because your presidency would create a conflict of interest between your constitutional role as Head of State and your status as an accused in a matter that has been brought against you by the State itself."
The NPA has 18 charges pending against Zuma.
"If you do not stand trial on these charges, a pall of suspicion will hang over every move you make and every decision you take in the future."
Zille said Zuma might have noticed that in mature democracies, unresolved allegations of impropriety sent politicians into early, and sometimes temporary, retirement.
"Yet, you have chosen to pursue your political career (and presidential ambitions), despite the fact that you have not publicly dealt with the serious and varied allegations the prosecution thinks it can prove against you in a court of law.
"Your trial must go ahead. Once the criminal proceedings are finally over, you can re-enter the world of politics, unless any sentence imposed precludes you from doing so.
"However, until then, you are trying to play for both sides at the same time. You can't. Nobody can. It's illegal and irrational.
"Any vote in Parliament in favour of your election as president is open to legal challenge on the basis that it would be invalid for want of consistency with the constitution," Zille said.
It was also politically and morally indefensible for Zuma to persist in his candidacy under the current circumstances.
"Presidents have to work in the political world of perceptions as well as the legal world of facts and rules. And so you must do the right thing and step aside until your innocence is proven in a court of law," she said.
Sapa