Background: Greater time spent sedentary is linked with increased risk of breast, colorectal, ovarian, endometrial, and prostate cancers. Given steadily increasing rates of mobile phone ownership, mobile phone interventions may have the potential to broadly influence sedentary behavior across settings. Objective: The purpose of this study was to examine the short-term impact of a mobile phone intervention that targeted sedentary time in a diverse community sample. Methods: Adults participated in a quasi-experimental evaluation of a mobile phone intervention designed to reduce sedentary time through prompts to interrupt periods of sitting. Participants carried mobile phones and wore accelerometers for 7 consecutive days. Intervention participants additionally received mobile phone prompts during self-reported sitting and information about the negative health impact of prolonged sedentariness. The study was conducted from December 2012 to November 2013 in Dallas, Texas. Linear mixed model regression analyses were conducted to evaluate the influence of the intervention on daily accelerometer-determined estimates of sedentary and active time. Results: Participants (N=215) were predominantly female (67.9%, 146/215) and nonwhite (black: 50.7%, 109/215; Latino: 12.1%, 26/215; other: 5.6%, 12/215). Analyses revealed that participants who received the mobile phone intervention had significantly fewer daily minutes of sedentary time (B=–22.09, P=.045) and more daily active minutes (B=23.01, P=.04) than control participants. Conclusions: A simple mobile phone intervention was associated with engaging in less sedentary time and more physical activity. Findings underscore the potential impact of mobile phone interventions to positively influence sedentary behavior and physical activity.