We aimed to investigate the expression of selenocysteine (Sec)-containing selenoproteins in natural Lactococcus lactis and to analyze the differences in glutathione peroxidase (GPx) activity among the recombinant L. lactis NZ9000/pNZ8148-GPx (NG1) and mutants specifically producing selenoproteins generated by introducing the stop codon UGA (encoding Sec) and the cis-acting selenocysteine insertion sequence (SECIS) into the three cysteine sites (C36, C63 and C81) and the penultimate lysine site (L156) of LlGPx (the glutathione peroxidase expressed by NG1) under non-induced conditions, under nisin induction and under Se-enriched conditions. The results showed that under Se-enriched conditions, the activity of LlGPx (89.10 mU/mg) was comparable to that of the mutants (56.17-84.45 mU/mg). NG1 showed a band of 17.8 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), while none of the mutants showed complete or truncated bands corresponding to selenoproteins. This suggested that introduction of UGA and SECIS alone was insufficient for efficient read-through to detectable levels in L. lactis NZ9000 under the experimental conditions. This study lays a foundation for the construction of novel selenoproteins and their production in lactic acid bacterial cell factories.