Cancer cells upregulate their glucose utilisation via aerobic glycolysis, in a phenomenon known as the Warburg effect. Akt signalling is a major driver of malignancy, including the Warburg effect. Therapeutic targeting of Akt often leads to reduced glucose uptake, which is often considered a pharmacodynamic biomarker and mediator of the anti-tumour effects of Akt inhibition. However, it has become increasingly apparent that cancer cell metabolism is far more complex and heterogenous. Of note, the glycolytic end product lactate, which is often present in tumours at elevated concentrations along with increased acidity, can be transported via monocarboxylate transporter 1 (MCT1) and used as a fuel by cancer cells. Therefore, we hypothesised that lactate utilisation induces resistance to Akt inhibitors that target glucose utilisation. We used the Akt inhibitor uprosertib, which reduces glucose uptake in patients, to investigate the influence of lactic acid on response to treatment in HCT116 and LS174T colon cancer cells in vitro. Stable isotope labelling and metabolomics were used to investigate lactic acid utilisation and its influence on growth and survival of cells. Results showed that lactic acid induced resistance to uprosertib treatment in HCT116 and LS174T cells. Furthermore, preferential lactate utilisation via the TCA cycle and enhanced oxidation was maintained in the presence of uprosertib treatment. Combining uprosertib with the mitochondrial Complex I inhibitor metformin or the MCT1 inhibitor AZD3965, was sufficient to re-sensitise cells to uprosertib treatment. Although lactic acid did not induce resistance to all Akt inhibitors, a kinase inhibitor screen identified many other inhibitors that showed a differential response in the presence of lactic acid. Overall, this study showed that lactic acidosis may be a novel mechanism of resistance to uprosertib and other kinase inhibitors and targeted to improve therapeutic response in patients.