Social media platforms are important channels through which health education about the utility and safety of vaccination is conducted. To investigate if tweets with different sentiments toward vaccination and different contents attract different levels of Twitter users’ engagement (retweets). A stratified random sample (N= 1425) of 142,891# vaccine tweets (February 4, 2010, to November 10, 2016) was manually coded. All 201 tweets with 100 or more retweets from 194,259# vaccineswork tweets (January 1, 2014, to April 30, 2015) were manually coded. Regression models were applied to identify factors associated with retweet frequency. Among# vaccine tweets, provaccine tweets (adjusted prevalence ratio= 1.5836, 95% confidence interval= 1.2130-2.0713, p< 0.001) and antivaccine tweets (adjusted prevalence ratio= 4.1280, 95% confidence interval= 3.1183-5.4901, p< 0.001) had more retweets than neutral tweets. No significant differences occurred in retweet frequency for content categories among antivaccine tweets. Among 411 links in provaccine tweets, Twitter (53; 12.9%), content curator Trap. it (14; 3.4%), and the Centers for Disease Control and Prevention (8; 1.9%) ranked as the top 3 domains. Among 325 links in antivaccine tweets, social media links were common: Twitter (44; 14.9%), YouTube (25; 8.4%), and Facebook (10; 3.4%). Among highly retweeted# vaccineswork tweets, the most common theme was childhood vaccinations (40%; 81/201); 21% mentioned global vaccination improvement/efforts (42/201); 29% mentioned vaccines can prevent outbreaks and deaths (58/201). Engaging social media key opinion leaders to facilitate health education about vaccination in their tweets may allow reaching a wider audience online.