Talks between the Rail Maritime and Transport Workers union (RMT) and Southern broke down last week in the 10-month-long dispute.
It centres on Southern's plan to make conductors on-board supervisors, giving drivers control of carriage doors.
Southern said it expects to run "around three-quarters of its normal service", despite the industrial action.
The rest will either be cut completely, be replaced by bus services, or run with fewer carriages than normal.
Live updates: Southern strike and Sussex news
Angie Doll, passenger services director at Southern said: "We will be running as many trains as possible on Wednesday but our advice to passengers is to expect some disruption and check on our website before you travel."
The 24-hour strike, which began at 00:01 GMT, comes after ongoing talks ended on 14 February without deal.
Two days later, members of train drivers' union Aslef voted against a deal union leaders had agreed with Southern.
A fresh set of talks with the rail firm got under way, earlier.
An Aslef spokesman said it was re-opening of negotiations at the earliest opportunity since the ballot result, but given the length of previous debates, a quick resolution was not expected.
Southern's parent firm Govia Thameslink (GTR) added: "All we can say is that lines of communication are open."
Guards on Southern trains have walked out on strike for the 29th time in their long-running row over safety.