Sea-ice microbial communities are integral to primary and secondary production in ice-covered regions of the Southern Ocean, but few studies have characterised the heterogeneity of microbes within the ice or determined whether habitat variability influences community dynamics. We examined the response of sea-ice microbes to key physicochemical variables by conducting an 18 d reciprocal transplant experiment within Antarctic fast-ice. A series of ice cores were extracted from 2.6 m annual ice and reinserted upside down to expose resident microbial assemblages to significantly different light, temperature and salinity regimes. The abundance and community composition of bacteria, microalgae and protozoa was subsequently determined within 3 sections of each core (top, middle and bottom) and compared with experimental controls. Results demonstrate that ice-associated microbes are finely attuned to discrete microhabitats within the sea-ice matrix. Positive growth and a shift in community composition was observed for microalgae moved from the top to the bottom of the ice, but significant bleaching of photosynthetic pigments resulted in zero net growth for bottom-ice communities exposed to the surface. Although bacteria may have been less vulnerable to initial change in their microenvironment, there was no significant increase in the average abundance of cells at either end of the flipped cores after 18 d, despite a presumed increase in algal-derived dissolved organic matter. This suggests a significant lag in the response time of bacteria to available growth substrates and a temporary ‘malfunction’ of the microbial loop.