The use of biofuel is essential for the reduction of greenhouse gas emission. This study highlights the use of biodiesel as a means of reducing greenhouse gas emission from the diesel engine of heavy-duty vehicles. Biodiesel is fatty acid methyl ester (FAME) obtained through ester exchange reaction by adding methanol to oil, such as rapeseed oil, soybean oil, palm oil, etc. The CO2 emission from combustion of biodiesel is defined to be equivalent to the CO2 volume absorbed by its raw materials or plants in their course of growth. On the other hand, however, operation of diesel engine with biodiesel is known to increase the NOx emission when compared with that with conventional diesel fuel. Then suppressing this NOx increase is regarded as a critical issue.This paper consists of two parts: comprehending the factors of NOx emission increase and improving this emission performance in a diesel engine fuelled with biodiesel. In the first part, the basic combustion and emission characteristics of biodiesel were analyzed by using a single-cylinder diesel engine with applying double and single injection. From the results of single-cylinder engine bench test, NOx emission from biodiesel operation was increased and smoke emission was decreased compared with those from conventional diesel operation. In addition, the factor of NOx emission increase was considered in detail using a zero-dimensional simple cycle simulation model. As a result, the factor of NOx increase in biodiesel seems to be changing of local excess air ratio in fuel spray compared with conventional diesel fuel.In the second part, NOx emission from biodiesel engine was decreased by the control of air entrainment in the fuel spray with optimization of fuel injection pressure and diameter of injection nozzle hole. From the results, making diameter of nozzle hole large and decreasing fuel injection pressure had a great effect on NOx reduction with suppressing smoke emission under medium load conditions.