The RMT revealed it had received a legal challenge just hours before the start of a three-day walkout.
Southern said in a statement it was "considering all possible options to stop the strike and that includes a legal challenge".
Talks aimed at averting the strikes over changes to the role of conductors ended without agreement last week.
The RMT said it intended to continue with the planned action but was examining the paperwork.
In a message to union members, RMT general secretary Mick Cash said: "The dispute is not over and the strike action... is definitely on.
"Organise the picket lines and protests and continue to show your determination and solidarity in your fight for justice."
The strike is due to begin from one minute past midnight on Tuesday, causing fresh travel misery for passengers.
In talks last week, the RMT said the train operator had rejected a fresh set of proposals that could have resolved the dispute.
But Govia Thameslink CEO Charles Horton said the RMT counter-proposal "didn't come anywhere near our goals of modernising our train service for the benefit of passengers".
RMT general secretary Mick Cash said: "RMT can confirm the receipt of a formal legal challenge from Southern Rail in the guards safety dispute.
"It is appalling that rather than sitting down with us at Acas today to seek a resolution the company has chosen to run to the courts under the cloak of the anti-union laws."
On Sunday, it emerged rail staff had been advised by union bosses to accept the new contracts.
The RMT said there was "no climb down" but it was their duty to protect staff against mass sackings.
A Southern spokesman said: "On Friday they told their conductor members to accept a deal and then tomorrow they plan to strike against it.
"It is a situation which will leave our passengers baffled and in that context we are presently considering all possible options to stop the strike.
"We have written to the RMT about the validity of the dispute in light of recent developments.
"We await their response and will consider all possible options to stop the strike and that includes a legal challenge."
Southern is taking legal action to try to stop a series of strikes from Tuesday, the RMT transport union said.