Using biodiesel fuel in diesel engines for heavy-duty transport is important to meet the stringent emission regulations. Biodiesel is an oxygenated fuel and its physical and chemical properties are close to diesel fuel, yet there is still a need to analyze and tune the fuel injection parameters to optimize the combustion process and emissions. A four-injections strategy was used: two pilots, one main and one post injection. A highly advanced SOI decreases the NOx and the compression work but makes the combustion process less efficient. The pilot injection fuel mass influences the combustion only at injection close to the top dead center during the compression stroke. The post injection has no influence on the compression work, only on the emissions and the indicated work. An optimal injection strategy was found to be: pilot SOI 19.2 CAD BTDC, pilot injection fuel mass 25.4%; main SOI 3.7 CAD BTDC, main injection fuel mass 67.3% mg; post SOI 2 CAD ATDC, post injection fuel mass 7.3% (the injection fuel mass is given as a percentage of the total fuel mass injected). This allows the indicated work near the base case level to be maintained, the pressure rise rate to decrease by 20% and NOx emissions to decrease by 10%, but leads to a 5% increase in PM emissions.