Low-temperature combustion has the potential to reduce nitrogen oxides (NOx) and particulate matter (PM) emissions; however, it faces the challenge of high level of hydrocarbon (HC) and carbon monoxide (CO) emissions. Application of oxygenated fuel such as biodiesel to LTC has gained interest to overcome this drawback. This study aims to provide a comprehensive understanding of the influence of biodiesel on premixed compression ignition (PCI) in a single-cylinder compression ignition engine. A series of engine tests were carried out at an engine speed of 1200 rev/min with two injection pressures (800 bar and 1600 bar), two injection timings (25 and 5 crank angle degree (CAD) before top dead center (bTDC)), and four exhaust gas recirculation (EGR) rates (0-30%). Engine performance results showed that fuel consumption with PCI combustion was higher than that with conventional diesel combustion (CDC) due to the advanced combustion phase. The lower heating value of biodiesel resulted in approximately 10% higher fuel consumption than diesel regardless of test conditions. In terms of emissions, PCI combustion indicated higher carbon CO and HC emissions because of liquid fuel flow in squish areas with early injection timing supported by a Lagrangian spray simulation. However, the application of biodiesel in PCI combustion was beneficial to suppress CO and HC emissions while maintaining low levels of NOx and PM emissions compared to CDC condition. Especially, the PN emission under PCI biodiesel condition was 1/10 of CDC. Soot morphology by TEM imaging showed a smaller primary particle diameter with biodiesel, attributed to enhanced soot oxidation.