Metabolomics can be used to identify the changes and metabolic pathways of nutrients and flavor substances in foods at specific processing or fermentation stages. This study revealed the accumulation and degradation of phytochemicals during the fermentation of kiwifruit juice (KJ) by lactic acid bacteria (LAB) through widely targeted metabolomics and explored the impact of different fermentation strategies on the nutritional quality and functional characteristics of KJ. LAB utilized the original sugars, acids, amino acids, and nucleotides of the matrix to produce numerous bioactive compounds, including phenols, organic acids, free fatty acids, and vitamins, while achieving bacterial proliferation and reduced anti-nutritional factors such as alkaloids, thus improving the nutritional and functional properties of KJ. Moreover, compared with monoculture fermentation, mixed fermentation promoted the production of more biologically active phenols and alkaloids, among which vanillic acid and luteolin-8-C-arabinoside were the characteristic metabolites of mixed and mono-fermentation. Subsequently, the metabolic pathways of key metabolites were predicted, including the glycolytic Embden-Meyerhof-Parnas pathway, pentose phosphoketolase pathway, organic acid metabolic and arginine deiminase pathways for improving the acid resistance of LAB, and the phenolic-acid-based phenylalanine and tyrosine metabolic pathway.