Background <p>Microhabitat environmental factors (e.g., temperature, oxygen concentration, nutrients, osmotic stress, and topography) are critical to the survival of intertidal organisms. Understanding how transcription responses are regulated in relation to intertidal microhabitat variation has important implications for studying adaptive evolution in these organisms. The barnacle <i>Chthamalus challengeri</i>, which survives in the intertidal zone and is subjected to periodic tidal changes, serves as an ideal species for detecting adaptive evolution in intertidal organisms.</p> Results <p>In this study, we designed a series of in situ tidal conditions for <i>C. challengeri</i> and sequenced their transcriptome collected from various microhabitats. We aimed to detect the genetic adaptation mechanisms of barnacles responding to the microhabitat changes in the intertidal zone based on comparative transcriptomics. Our results indicated that different intertidal microhabitats significantly affected the gene expression models of <i>C. challengeri</i>, particularly for genes related to physiological and biochemical functions. Specifically, the expression of genes such as CYP450, HSP70, CYTB, and COX1 was significantly increased under low tide (air-exposed conditions), while genes like CNGA3, AK, and CP52 showed significantly increased expression under high tide (seawater-immersed conditions).</p> Conclusion <p>The results suggest that <i>C. challengeri</i> relies on cytochrome p450 enzymes to enhance oxidative capacity, counts on heat shock proteins and cell phagocytosis to resist microhabitat changes in response to different tidal conditions, and produces a hypoxic stress response to regulate energy metabolism and body temperature changes upon entering into seawater. This study provides genetic resources and clues for investigating the adaptation mechanisms of intertidal barnacles and identifies different gene expression models for <i>C. challengeri</i> responding to various microhabitats.</p>