Background: Transportation costs can be a barrier to healthcare services, especially for low-income, disabled, elderly, and geographically isolated populations. This study aimed to estimate the transportation costs of healthcare service utilization and related influencing factors in Korea in 2016. Methods: Transportation costs were calculated using data from the 2016 Korea Health Panel Study. A total of 14,845 participants were included (males, 45.07%; females, 54.93%), among which 2,148 participants used inpatient and 14,787 used outpatient care services. Transportation costs were estimated by healthcare types, transportation modes, and all disease and injury groups that caused healthcare service utilization. The influencing factors of higher transportation costs were analyzed using multivariable regression analysis. Results: In 2016, the average transportation costs were United States dollars (USD) 43.70 (purchasing power parity [PPP], USD 32.35) per year and USD 27.67 (PPP, USD 20.48) per visit for inpatient care; for outpatient case, costs were USD 41.43 (PPP, USD 30.67) per year and USD 2.09 (PPP, USD 1.55) per visit. Among disease and injury groups, those with neoplasms incurred the highest transportation costs of USD 9.73 (PPP, USD 7.20). Both inpatient and outpatient annual transportation costs were higher among severely disabled individuals (inpatient, +USD 44.71; outpatient, +USD 23.73) and rural residents (inpatient, +USD 20.40; outpatient, +USD 28.66). Transportation costs per healthcare visit were influenced by healthcare coverage and residential area. Sex, age, and income were influencing factors of higher transportation costs for outpatient care. Conclusion: Transportation cost burden was especially high among those with major noncommunicable diseases (e.g., cancer) or living in rural areas, as well as elderly, severely disabled, and low-income populations. Thus, there is a need to address the socioeconomic disparities related to healthcare transportation costs in Korea by implementing targeted interventions in populations with restricted access to healthcare.