Members of the Rail, Maritime and Transport (RMT) union are staging a 48-hour walkout, which ends at midnight on Saturday, hitting services across southern England, including the Portsmouth line.
More action is planned in the coming weeks, and the union has escalated the row by calling strikes over Christmas and the New Year.
RMT members mounted picket lines at stations including Brighton and London Victoria.
Southern condemned the latest stoppage as ‘spiteful and vindictive’ and warned of disruption to services even though it planned to run more trains than during previous strikes.
The bitter dispute over the role of conductors is completely deadlocked.
RMT general secretary Mick Cash said: ‘Despite a vicious campaign of company bullying, threats and intimidation, the strike action on Southern this morning remains absolutely rock-solid.
‘Passengers know that Southern Rail under GTR is an unmitigated disaster that puts profits before public safety. That is the issue at the core of this dispute.
‘The solution to this dispute can be achieved easily through a cast-iron guarantee of a second, safety-critical member of staff on all current services with a guard.
‘Once again this morning the union is demanding that Transport Secretary Chris Grayling gets out of his bunker, gets hold of this basket-case company and gets round the table for serious negotiations.’
A Southern spokesman said: ‘These strikes are spiteful, vindictive and pointless given the majority of conductors have now signed up to the new on-board role.
‘This has never been about safety - it’s purely about the RMT hanging on to outdated working practices and union power.’
The union accused the company and the government of “sheer pig-headedness” as it announced that its members will strike from 0001 on December 22 until 2359 on Christmas Eve and again from 0001 on New Year’s Eve until 2359 on January 2, when new fare rises come into effect.
No trains will stop tomorrow at Lewes, where tens of thousands of people usually attend the town’s bonfire.