Lactate-rich byproducts and fermentation residues are generated during various food and fermentation processes, presenting opportunities for their conversion into value-added products through microbial bioprocesses. Aspergillus oryzae is a promising microbial host for such applications because of its ability to use diverse substrates and produce valuable products. However, the ability of A. oryzae to assimilate lactate and the factors limiting this process remain poorly understood despite its industrial importance. In this study, we investigated the transcriptional responses of A. oryzae during lactate assimilation and evaluated the culture conditions promoting this process. A. oryzae demonstrated only limited growth and lactate consumption when lactate was provided as its only carbon source in the synthetic minimal medium. Transcriptome analysis suggested possible metabolic constraints associated with carbon flux into the tricarboxylic acid (TCA) cycle, limiting lactate assimilation. To alleviate these constraints, the culture conditions were examined. Supplementation with small amounts of sugars, specific amino acids, and α-ketoglutarate promoted lactate assimilation. Moreover, low-pH conditions further improved lactate assimilation, reducing residual lactate concentrations to below the detection limit. These findings indicate that efficient lactate assimilation is promoted by small amounts of metabolic co-substrates, potentially through effects on central carbon metabolism, together with favorable low-pH conditions.