Health literacy is important for health behavior engagement. Therefore, it is important to have a good instrument assessing health literacy with a theoretical framework. The present study aimed to examine the measurement invariance and differential item functioning (DIF) of a newly developed health literacy instrument; that is, the Health Literacy Instrument for Adults (HELIA). Confirmatory factor analysis (CFA) and Rasch models were used to examine the data collected from a large Iranian sample (N = 9678; 67.3% females; mean age = 36.44 years). All the participants completed the HELIA. CFA was used to examine if the HELIA had a five-factor structure (including reading, access to information, understanding, appraisal, and decision making/behavioral intention factors) and multigroup CFA to examine if the five-factor structure of HELIA was invariant across gender, educational level, accommodation, and age subgroups. Rasch models were used to examine whether each factor of HELIA was unidimensional and DIF contrast in Rasch to examine if the HELIA items were interpreted similarly across the aforementioned subgroups. The CFA results supported the five-factor structure of HELIA, and the Rasch models verified that each HELIA factor is unidimensional. Additionally, multigroup CFA supported the measurement invariance of HELIA across the following subgroups: male vs. female; highly educated vs. poorly educated; city residents vs. suburban residents; and younger age vs. older age. The DIF contrasts in the Rasch models additionally showed that there are no substantial DIF items in the HELIA across aforementioned subgroups. Therefore, the HELIA is a feasible and comprehensive instrument assessing health literacy across different populations in Iran.