OBJECTIVE: We examined whether teleworking preference and frequency were associated with work functioning impairment. METHODS: This online cross-sectional study was conducted using a self-administered questionnaire among 27,036 full-time Japanese workers. The Work Functioning Impairment Scale was used to measure work functioning impairment, and we performed multilevel logistic regression analysis. RESULTS: Higher odds ratios for work functioning impairment were observed among employees who preferred to telework compared with those who preferred working in the workplace. A similar trend was observed among employees who teleworked 4 or more days a week compared with those who almost never teleworked. When teleworking preference and frequency were adjusted, only teleworking preference was associated with work functioning impairment. CONCLUSIONS: A preference for teleworking was associated with work functioning impairment and one factor that increased the teleworking frequency.