Based on the measured injection rates obtained from the spray momentum experiment, the three-dimensional computational fluid dynamics simulation study on the effect of injection rate from each nozzle hole on spray characteristics and combustion process was conducted for a one-cylinder diesel engine. The simulation model was successfully verified by the data of the experiment. The results show that at the beginning and mid-stages of injection, the nozzles with a higher transient injection rate exhibit higher jet velocity, bigger spray penetration distance, and wider equivalence ratio distribution. Besides, the disturbance induced by fuel injection on their surrounding gas is higher. Due to the difference in injection rates from each nozzle hole in the cylinder, gas–fuel mixtures are non-uniform. In the case of measured injection rates from each nozzle hole, Hole 4 records the highest instantaneous injection rate. This results in the injection of more fuel during ignition delay. More heat generated from thermal chain reactions raises fuel spray temperatures and quicker ignition of mixtures. In the case of uniform simulated injection rate (injection quantity values are the same as in the previous case), more uniform flow fields and stronger small swirl motions were generated that enhance fuel atomization and mixture formations. At the later stages of injection and combustion, quicker diesel fuel burning rate with a centralized exothermic reaction process occurs due to in-cylinder uniform fuel distribution and air motion. In the case of simulating uniform injection rate from three holes and non-injection from one (same injection quantity values as previous cases), uneven fuel distribution that occurs in the cylinder will result in poor mixture formations and subsequently poor combustion, and more afterburning will occur.