These last years, much of researches were carried out to find the appropriate substitution fuel to the fossil fuels. The use of biofuel prepared from non-edible vegetable oils are becoming a promising source to produce a fuel for diesel engine, commonly referred to as “biodiesel”. Considering the high oil extraction yield (around 40%) and the great quantity of pistacia lentiscus (PL) trees available in arid and semi-arid areas of Mediterranean countries, it is selected in the present work to study the biodiesel prepared from PL oil. PL biodiesel is obtained by converting PL seed oil with a single-step homogenous alkali catalyzed transesterification process. PL biodiesel characterization, according to the standard methods, shows that the physicochemical properties are comparable to those of conventional diesel fuel. In a second part, a single cylinder air-cooled, DI diesel engine is used to test PL biodiesel at 1500 rpm under various engine load conditions. The combustion characteristics (cylinder pressure, ignition delay, heat release rate and combustion duration) of biodiesel are compared with those of diesel fuel. The combustion characteristic results show in all engine load conditions that biodiesel exhibits shortens ignition delays with extended combustion duration.