Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis. •Lactococcus lactis is a useful alternative to E. coli for expressing toxic proteins such as HIV-1 Tat.•P2 promoter is the best choice among PepN, NisA, P11, P170 and P2 promoters for HIV-1 Tat expression.•Expression through the P2 promoter demonstrates the least proteolytic processing in the MG1363 strain of L. lactis.•0.25% Glucose without pH adjustment serves as the best nutritional condition for HIV-1 Tat expression.