BACKGROUND: Tobacco use remains the leading preventable cause of morbidity and mortality in the United States. Novel interventions are needed to improve smoking cessation rates. Mindfulness-based interventions (MBIs) for cessation address tobacco use by increasing awareness of the automatic nature of smoking and related behaviors (eg, reactivity to triggers for smoking) from a nonjudgmental stance. Delivering MBIs for smoking cessation via innovative technologies allows for flexibility in the timing of intervention delivery, which has the potential to improve the efficacy of cessation interventions. Research shows MBIs target key mechanisms in the smoking cessation process and can be used to minimize drivers of smoking lapse. OBJECTIVE: This single-arm study investigated the impact of mindfulness-based strategies and motivational messages on proximal outcomes, collected via ecological momentary assessment (EMA), relevant to tobacco abstinence via a microrandomized trial. This approach allows for the evaluation of intervention content on proximal outcomes (eg, reduced negative affect) that are thought to impact positive distal outcomes (eg, smoking abstinence). METHODS: All participants were motivated to quit smoking, and the intervention they received included nicotine replacement therapy, brief individual counseling, and a 2-week Just-in-Time Adaptive Intervention (JITAI) with wearable sensors. Throughout the JITAI period, a single strategy was randomly pushed (vs not) multiple times per day through the smartphone application. An EMA next assessed negative affect, positive affect, mindfulness, abstinence self-efficacy, motivation to quit, craving, and smoking motives. The primary analyses evaluated differences in EMA outcomes (proximal) for when a strategy was pushed versus not pushed. Additional analyses evaluated changes in similar outcomes collected from surveys at the baseline and end-of-treatment visits. RESULTS: Participants (N=38) were 63% (24/38) female, 18% (7/38) Hispanic or Latino, and 29% (11/38) African American. They had an average age of 49 years and smoked an average of 15 (SD 7.9) cigarettes per day. Results indicated that receiving the JITAI significantly reduced proximal negative affect in the second (and final) week of the intervention. Self-reports provided at baseline and end of treatment showed significant decreases in perceived stress, automaticity of smoking and craving, and a significant increase in abstinence self-efficacy. Increases in abstinence self-efficacy significantly predicted abstinence. CONCLUSIONS: To our knowledge, this is the first study to test the proximal impact of a mindfulness-based JITAI on key variables associated with smoking cessation. Our primary finding was that negative affect was lower following the completion of a strategy (vs when no strategy was delivered) in the final week of the JITAI. Among a larger sample size, future research should extend the length of the intervention to further evaluate the impact of the JITAI, as well as include a comparison condition to further evaluate how each component of the intervention uniquely impacts outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT03404596; https://clinicaltrials.gov/study/NCT03404596.