Objective: The aim of this study was to assess associations of various content areas of Twitter posts with help-seeking from the US National Suicide Prevention Lifeline (Lifeline) and with suicides. Methods: We retrieved 7,150,610 suicide-related tweets geolocated to the United States and posted between 1 January 2016 and 31 December 2018. Using a specially devised machine-learning approach, we categorized posts into content about prevention, suicide awareness, personal suicidal ideation without coping, personal coping and recovery, suicide cases and other. We then applied seasonal autoregressive integrated moving average analyses to assess associations of tweet categories with daily calls to the US National Suicide Prevention Lifeline (Lifeline) and suicides on the same day. We hypothesized that coping-related and prevention-related tweets are associated with greater help-seeking and potentially fewer suicides. Results: The percentage of posts per category was 15.4% (standard deviation: 7.6%) for awareness, 13.8% (standard deviation: 9.4%) for prevention, 12.3% (standard deviation: 9.1%) for suicide cases, 2.4% (standard deviation: 2.1%) for suicidal ideation without coping and 0.8% (standard deviation: 1.7%) for coping posts. Tweets about prevention were positively associated with Lifeline calls (B = 1.94, SE = 0.73, p = 0.008) and negatively associated with suicides (B = −0.11, standard error = 0.05, p = 0.038). Total number of tweets were negatively associated with calls (B = −0.01, standard error = 0.0003, p = 0.007) and positively associated with suicide, (B = 6.4 × 10−5, standard error = 2.6 × 10−5, p = 0.015). Conclusion: This is the first large-scale study to suggest that daily volume of specific suicide-prevention-related social media content on Twitter corresponds to higher daily levels of help-seeking behaviour and lower daily number of suicide deaths.