Biodiesel has emerged as a sustainable renewable transport fuel worldwide. This study investigated the use of biodiesel (B100) and mineral diesel in two identical unmodified vehicles to realistically assess different aspects of biodiesel’s compatibility with modern common rail direct injection (CRDI) diesel engines. Exhaustive experimental results from this series of tests are divided into four segments and no-load engine speed emissions, highway driving emissions, and acceleration characteristics of biodiesel and diesel-fueled vehicles are included in this first paper of the series of four papers. First, the emissions were measured at different engine speeds at no load, when the vehicles were stationary. Thereafter, the real highway driving emissions were measured at different vehicle speeds using on-board emission analyzers. CO and HC emissions from biodiesel fueled stationary vehicle were negligible but relatively higher than mineral diesel-fueled stationary vehicle. NOx emissions increased with increasing engine speed at no-load condition for both stationary vehicles and smoke opacity was also very low for most engine speeds at no load. Among real highway driving emissions, CO and HC from biodiesel-fueled vehicles were ∼13% and ∼30% lower than mineral diesel-fueled vehicles, respectively, at varying vehicle speeds. Emissions of CO2 were not too different in these two vehicles but NOx emissions from biodiesel-fueled vehicle were ∼32% higher than mineral diesel-fueled vehicle. Smoke opacity from biodiesel-fueled vehicle was lower by ∼80% than mineral diesel-fueled vehicle and it didn’t increase with increasing vehicle speed. Acceleration characteristics of biodiesel-fueled vehicle were as good as diesel-fueled vehicle up to a speed of 90 kmph using stock electronic control unit (ECU), however, beyond 90 kmph vehicle speed, the acceleration of biodiesel-fueled vehicle was noticeably lower than baseline mineral diesel-fueled vehicle. This underlines the need for ECU recalibration before the large-scale implementation of biodiesel in existing diesel vehicles.