Background Existing smoking cessation treatments are challenged by low engagement and high relapse rates, suggesting the need for more innovative, accessible, and interactive treatment strategies. Twitter is a Web-based platform that allows people to communicate with each other throughout the day using their phone. Objective This study aims to leverage the social media platform of Twitter for fostering peer-to-peer support to decrease relapse with quitting smoking. Furthermore, the study will compare the effects of coed versus women-only groups on women’s success with quitting smoking. Methods The study design is a Web-based, three-arm randomized controlled trial with two treatment arms (a coed or women-only Twitter support group) and a control arm. Participants are recruited online and are randomized to one of the conditions. All participants will receive 8 weeks of combination nicotine replacement therapy (patches plus their choice of gum or lozenges), serial emails with links to Smokefree.gov quit guides, and instructions to record their quit date online (and to quit smoking on that date) on a date falling within a week of initiation of the study. Participants randomized to a treatment arm are placed in a fully automated Twitter support group (coed or women-only), paired with a buddy (matched on age, gender, location, and education), and encouraged to communicate with the group and buddy via daily tweeted discussion topics and daily automated feedback texts (a positive tweet if they tweet and an encouraging tweet if they miss tweeting). Recruited online from across the continental United States, the sample consists of 215 male and 745 female current cigarette smokers wanting to quit, aged between 21 and 59 years. Self-assessed follow-up surveys are completed online at 1, 3, and 6 months after the date they selected to quit smoking, with salivary cotinine validation at 3 and 6 months. The primary outcome is sustained biochemically confirmed abstinence at the 6-month follow-up. Results From November 2016 to September 2018, 960 participants in 36 groups were recruited for the randomized controlled trial, in addition to 20 participants in an initial pilot group. Data analysis will commence soon for the randomized controlled trial based on data from 896 of the 960 participants (93.3%), with 56 participants lost to follow-up and 8 dropouts. Conclusions This study combines the mobile platform of Twitter with a support group for quitting smoking. Findings will inform the efficacy of virtual peer-to-peer support groups for quitting smoking and potentially elucidate gender differences in quit rates found in prior research. Trial Registration ClinicalTrials.gov NCT02823028; https://clinicaltrials.gov/ct2/show/NCT02823028 International Registered Report Identifier (IRRID) DERR1-10.2196/16417