In this study, mixed convection heat transfer characteristics of nanofluid flow in a circular minichannel were investigated experimentally. The effects of the particle volume ratio (0, 0.25, and 0.75%) and Reynolds number (200-60) on heat transfer by mixed convection were investigated for aiding and opposing flow conditions. Water and water based SiO2 nanofluids were used as working fluid in a minichannel with a diameter of 1.9 mm, and constant heat flux was applied to the outer surface of the minichannel. Temperature-dependent thermophysical properties such as thermal conductivity and viscosity, obtained by experimental measurements, were used in heat transfer calculations. Pressure based numerical computations for all experimental cases were also made by using single phase approach. The results were analyzed separately for aiding flow condition in which secondary flows originating from natural convection are in the same direction with the forced flow, and for opposing flow condition in which secondary flows are in the opposite direction with forced flow. For detailed analysis of mixed convection heat transfer, temperature contours and velocity profiles were obtained by the numerical computations which were compared and validated with the experimental results. According to the data obtained, it was determined that the addition of nanoparticles into the pure water increased the Nusselt number by around 21–64% for the aiding flow, and 18–58% for the opposing flow. The heat transfer in the aiding flow was observed to be minimum 4% and maximum 16% higher in comparison with the opposing flow condition. It was concluded that the direction of secondary flows significantly affects heat transfer.