Calcite-rich columnar stromatolites grew in perennially ice-covered Lake Joyce in the McMurdo Dry Valleys, Antarctica, during a period of environmental change associated with rising lake level. Stromatolite calcite contains carbon and oxygen isotope records of changes to microbial activity in response to variable light environments and water chemistry through time. The stromatolites grew synchronously with correlative calcite zones. The innermost (oldest) calcite zone has a wide range of δ¹³Ccalcite values consistent with variable photosynthetic effects on local DIC ¹³C/¹²C. Subsequent calcite zones preserve a progressive enrichment in δ¹³Ccalcite values of approximately + 2.6‰ through time, with δ¹³Ccalcite values becoming less variable. This enrichment likely records the removal of ¹²C by photosynthesis from the DIC reservoir over decades, with photosynthetic effects decreasing as light levels became lower and more consistent through time. Mean δ¹⁸Ocalcite values of the innermost calcified zone were at least 1‰ lower than those of the other calcified zones (t test p-level < 0.001). The significant difference in δ¹⁸Ocalcite values between the innermost and other calcified zones could be a product of mixing source waters with different isotopic values associated with the initiation of lake stratification associated with rising lake level. Overall, Lake Joyce stromatolites record significant lateral variability in relative photosynthetic rate and long-lived lake water stratification with microbial modification of the DIC pool. Such processes provide criteria for interpreting microbial activity within polar paleolake deposits and may shed light on variability in lake environments associated with changing climate in the McMurdo Dry Valleys.