Objective The study explores the differences of injection behaviors and quality between diabetic patients using 5 mm and 8 mm insulin pen needles, and the influencing factors. Methods This survey is based on purposive sampling. Data are collected with a structured questionnaire, including demographic data, injection behaviors and quality, from patients in 11 diabetes health promotion institutions in Taiwan. Results There are 1027 subjects average age 51.4. Subjects with elementary and high school degrees rank first and second. The average durations of diabetes and insulin injection are 12.6 and 5.3 years. The average hemoglobin A1c is 8.3%. Significant differences are found between subjects and their demographic data, including age, education level, body mass index, duration of diabetes, insulin injection duration and frequency. The use of 5 and 8 mm needles is also significantly related to injection behaviors, such as injection site rotation, person giving insulin shots, pinching the skin, injection angle and lipodystrophy on injection sites. Magnificent differences are noticed among the use of 5 and 8 mm needles and injection accessibility, anxiety and convenience. Factors influencing lipodystrophy include age, injection duration, interval, angle and insulin leakage. Factors affecting anxiety degrees are gender, age, injection duration, repeated use of needle, pinching of skin and insulin leakage. Conclusion The results showed that subjects using 8 mm pen needle have more injection site rotation than 5 mm users. 5 mm pen needle users have higher rates in self-injection, lipodystrophy, pinching of skin on injection site and injection angle (45°) than 8 mm users. 5 mm needle users show better performance regarding injection convenience, accessibility and anxiety than 8mm users. This research aims to provide guidelines on insulin pen injection education for clinical and community nursing professionals, and help diabetic patients obtain treatment efficacy.