Abstract Objective This study explores dose–response relationships when treating fibromyalgia with low-dose naltrexone. Design A single-blinded clinical trial was carried out using the “up-and-down” method. Subjects Subjects included women with a diagnosis of fibromyalgia aged 18–60 years who had been referred to treatment at a public pain clinic at a Danish university hospital. Methods The test doses were in the range 0.75–6 mg, and the dosing interval was 0.75 mg. The method was sequential and allowed predicting the dose effective in 50% (ED50) and 95% (ED95) of the subjects when the dose had shifted direction 10 times, and six pairs of “up-and-down” data were available. Results A total of 27 subjects were included in the study; two subjects were withdrawn. After inclusion of 25 evaluable subjects, the dose estimates were calculated as 3.88 mg for ED50 and 5.40 mg for ED95. As a secondary outcome, the effects on 10 common fibromyalgia symptoms were evaluated. A high interindividual variation was observed both in the symptom presentation at baseline and in which symptoms were reduced by low-dose naltrexone. Conclusions This study is the first to explore dose–response relationships in the treatment of fibromyalgia with low-dose naltrexone. Future placebo-controlled randomized clinical trials are needed, and according to our findings, 4.5 mg, which has previously been used, seems to be a relevant test dose. We recommend that future studies include additional nonpain fibromyalgia symptoms as outcome measures.