A shop worker said she was "mortified" when she opened her locker and discovered a homophobic note.
The offensive message was found by Pammie Clinton during her shift at Wilko in Arnold, Nottinghamshire, on 9 September.
It directed a homophobic slur at her and implored her to quit. She handed in her notice a few days later.
Wilko said discrimination of any kind was not accepted and it was looking into the situation "very seriously".
Ms Clinton, who is a lesbian, said the message had been pushed into her locker through a small hole.
"I was absolutely disgusted with what I was reading - I felt like my heart had sunk to the pit of my stomach," she said.
"I went straight to the bathroom and sat and cried."
The incident was reported to management but the culprit was never found - and Ms Clinton said she felt compelled to resign.
She said: "I was filled with anxiety and found myself looking at everyone at work thinking 'was it you?', which was mentally and emotionally unhealthy."
Ms Clinton has since found work elsewhere but hopes her experience will lead to changes at Wilko.
"I hope management can deliver and enforce equality and diversity training to educate, accept and welcome everyone," she said.
"There are some incredibly kind, honest and innocent members of staff at Arnold. This is the mindset of one very narrow-minded homophobic individual.
"But we need to remember that lives have been lost because of homophobia - sometimes it just takes one 'flippant' comment for someone to no longer be able to tolerate it anymore."
A Wilko spokesperson said: "We welcome everyone at Wilko and want our customers and team members to feel comfortable being themselves.
"We think of ourselves as one family with strong values, one of which is respect. So, discrimination of any kind isn't accepted.
"However, sometimes even families can let each other down and if this happens we really want to put it right.
"We're sorry our team member felt they had to resign and we've already been looking into the situation very seriously.
"As a highly valued team member, we've also asked them to have another think about their resignation."