Set up by grocers' charity Caravan, the helpline will provide counsellors to assist retailers who have experienced incidents which were harmful, threatening or challenging.
They will also be able to discuss relationships, health and money problems.
"It's a support service tailor-made for each individual," Caravan welfare development director Cathy Mercer said.
"It's about helping people through difficult situations and providing a duty of care. It also minimises time lost through staff being away from work."
Baz Patel, owner of Molly's in Luton, supported the initiative. "Some staff go and stew at home, when they really need to talk to someone," he said. "They might not want to talk to their store manager about an issue, but it could certainly help to speak to a professional."
Caravan is currently helping 1,900 people, 30% of whom work or have worked for convenience stores. The new helpline number is 0808 802 0304.