Background Suicides and opioid overdose deaths are among the most pressing public health concerns in the US. However direct evidence for the association between opioid use and suicidal behavior is limited. The objective of this article is to examine the association between frequency and dose of prescription opioid use and subsequent suicide attempts. Methods and findings This retrospective cohort study analyzed 4 years of statewide medical claims data from the Connecticut All-Payer Claims Database. Commercially insured adult patients in Connecticut (n = 842,773) who had any medical claims beginning in January 2012 were followed through December 2015. The primary outcome was suicide attempt identified using International Classification of Diseases (ICD 9) diagnosis codes. Primary predictor variables included frequency of opioid use, which was defined as the number of months with claims for prescription opioids per year, and strength of opioid dose, which was standardized using morphine milligram equivalent (MME) units. We also controlled for psychiatric and medical comorbidities using ICD 9 codes. We used Cox proportional hazards regression to examine the association between frequency, dose, and suicide attempts, adjusting for medical and psychiatric comorbid conditions. Interactions among measures of opioid use and comorbid conditions were analyzed. In this cohort study with follow-up time up to 4 years (range = 2–48 months, median = 46 months), the hazard ratios (HR) from the time-to-event analysis indicated that patients prescribed opioid medications for at least 6 months during the past year and at 20–50 MME levels or higher had 4.44 (95% CI: [3.71, 5.32]) to 7.23 (95% CI: [6.22, 8.41]) times the risk of attempted suicide compared to those not prescribed opioids. Risk of suicide attempt was sharply elevated among patients with psychiatric conditions other than anxiety who were prescribed more frequent and higher opioid doses. In contrast, more frequent and higher doses of prescription opioids were associated with lower risk of suicide attempts among patients with medical conditions necessitating pain management. This study is limited by its exclusive focus on commercially insured patients and does not include patients covered by public insurance. It is also limited to patients’ receipt of prescription opioids and does not take into account opioids obtained through other means, nor does it include measures of actual patient opioid use. Conclusions This analysis provides evidence of a complex relationship among prescription opioids, mental health, pain and other medical comorbidities, and suicide risk. Findings indicate the need for proactive suicide surveillance among individuals diagnosed with affective or psychotic disorders who are receiving frequent and high doses of opioids. However, appropriate opioid treatment may have significant value in reducing suicide risk for those without psychiatric comorbidities.