The study investigates the effects of non-esterified rapeseed oil, a commonly used liquid first-generation renewable bio-fuel utilized in existing compression ignition engines, on the behavior of the fuel in the high-pressure diesel fuel injection system with a traditional mechanically controlled fuel injection pump. The experiments were carried on a Zetor tractor engine powered by diesel fuel, biodiesel and heated fuel-grade rapeseed oil, instrumented with direct and indirect fuel injection line pressure sensors. The order-of-magnitude higher viscosity, higher bulk modulus, and higher density of rapeseed oil compared to diesel fuel were found to have measurable effects on the fuel delivery timing, fuel pressure, load on the fuel injection pump, and maximum fuel delivery rates. The load on the fuel injection pump was increased, and the rapeseed oil was delivered earlier and at higher pressures, counteracting the poorer atomization and increased combustion delay, with differences decreasing with increasing fuel temperature.