In order to improve the utilization efficiency of copper oxide mineral resources, a novel collector, 5-hydroxyoctan-4-one oxime (HOO), was synthesized from a commodity chemical butyraldehyde and hydroxylamine hydrochloride. Its flotation performance and adsorption mechanism to malachite were investigated by flotation tests, Fourier transform infrared spectrum (FTIR), X-ray photoelectron spectroscopy (XPS) analysis and density functional theory (DFT) calculation. Flotation results of malachite showed that HOO exhibited better collecting ability and less dosage compared to benzohydroxamic acid (BHA). FTIR and XPS data gave clear evidence for the formation of Cu-oxime complex (five-membered and six-membered ring chelates) on malachite surfaces after HOO adsorption through the linkage among C—OH, C=NOH and Cu species. Moreover, DFT results showed that HOO coordinated with Cu atoms preferably through the oxime and hydroxyl groups to form bidentate chelates.