The use of biodiesel is becoming inevitable due to the depletion of fossil fuel resources. Biodiesel is an attractive alternative fuel derived from natural oils and can be used directly in diesel engines with no major change. However, various biodiesels may exhibit different performance behaviors and emission characteristics, with some performing worse than diesel fuel. The present research work investigates the performance, combustion, and emission behavior of rubber seed biodiesel (RSB)/diesel blends (B20, B30, B40) and B20 + ethyl hexyl nitrate (EHN) at four injection timings (19°, 21°, 25°, and 27° before top dead center [BTDC]) in a single-cylinder DI diesel engine. Combustion of biodiesel/diesel blends generally resulted in worse performance, except smoke emission. The addition of EHN reduced hydrocarbon (HC) emissions but negatively impacted brake-specific fuel consumption (BSFC) and brake thermal efficiency (BTE). However, advanced injection timing not only restored the combustion parameters to the B20 level but also brought them closer to those of the diesel engine. Advancing the injection timing to 27° BTDC improved BSFC and BTE by 3% and 4% compared to B20, respectively. Additionally, the HC emission decreased strongly by 80% and 73%, and smoke emission decreased by 15% and 16%, respectively, compared to B20 and diesel fuel values. A slight improvement in NOx emissions (by 2%) was also observed compared to B20. An increase in cylinder pressure from 66.2 to 67.4 bar was observed with advanced injection timing, contributing to improved engine performance. Analysing of combustion characteristics showed that RSB/diesel blends, when doped with EHN, offer better performance at advanced injection timings making them a suitable alternative fuel to replace diesel fuel usage in developing countries like India.