In this study, the effects of negative valve overlap (NVO) on homogenous charge compression ignition (HCCI) combustion and engine performance were experimentally investigated. A four stroke, single cylinder, port injection HCCI engine was operated at −16 deg crank angle (CA), −8 deg CA, and +8 deg CA valve overlap values and different lambda values and engine speeds at wide open throttle. RON40 and RON60 were used as test fuels in view of combustion and performance characteristics in HCCI mode. The variations of indicated mean effective pressure (IMEP), residual gas, CA50, indicated thermal efficiency (ITE), indicated specific fuel consumption (ISFC), maximum pressure rise rate (MPRR) and ringing intensity (RI) were observed on HCCI combustion. The results showed that NVO caused to trap residual gases in the combustion chamber. Hot residual gases showed heating and dilution effect on HCCI combustion. Combustion was retarded with the presence of residual gas at −16 deg CA NVO. Test results showed that higher imep and maximum in-cylinder pressure were obtained with RON60 according to RON40. As expected, CA50 was obtained later with RON60 compared to RON40 due to more resistance of auto-ignition. RON60 residual gas prevented the rapid and sudden combustion due to higher heat capacity of charge mixture. RI decreased with the usage of RON60 compared to RON40. Significant decrease was seen on RI with RON60 especially at lower lambda values. It was seen that HCCI combustion can be controlled with NVO and operating range of HCCI engines can be extended.