Abstract: This paper presents an experimental investigation of the hydraulic and thermal fields of a 29 nm CuO nanoparticle–water nanofluid with various volume fractions, 0.24%, 1.03% and 4.5% flowing inside a rectangular microchannel heat sink under both laminar and turbulent conditions. The isothermal and heated tests are conducted for Reynolds number up to ≈5000 and to ≈2500, respectively. For a given fluid flow rate experimental results show an increase of the pressure drop and the friction factor with respect to water. This increase can be as high as 70%, 25%, and 0–30%, respectively, for the 4.5%, 1.03%, and 0.24% particle volume fractions. Although the laminar-to-turbulent transition was observed at nearly the same critical Reynolds number Rec ≈ 1000 for water and the tested nanofluids, this value of Rec is clearly lower than that corresponding to a smooth surface microchannel. Results show a slight heat transfer enhancement with respect to water for nanofluids with low particle volume fractions, 0.24% and 1.03%, while for the 4.5% fraction a clear decrease of heat transfer was found. In general, the nanofluid overall energetic performance, defined by the heat transferred/pumping power ratio, remains lower than that of water for a given Reynolds number. This ratio decreases with an augmentation of the particle volume fraction.