•  The molecular basis of left–right asymmetries in brain structure and function is a central question in neuroscience. •  We have previously demonstrated that the neuronal circuitry composed of hippocampal pyramidal neurones is asymmetrical depending on the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. •  In this study, we analysed the hippocampus of β2‐microglobulin (β2m)‐deficient mice lacking stable cell surface expression of major histocompatibility complex class I (MHCI), which is known to be important in cellular immunity. •  We found that β2m‐deficient mice lacked structural and functional asymmetries in hippocampal circuitry, suggesting that MHCI is critical for the generation of hippocampal asymmetry. •  Our results provide a first step in elucidating the cellular process that generates brain asymmetries. Abstract  Left–right asymmetry is a fundamental feature of higher‐order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluRɛ2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of ɛ2 subunits affects the properties of NMDARs and generates two populations of synapses, ‘ɛ2‐dominant’ and ‘ɛ2‐non‐dominant’ synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of β2‐microglobulin (β2m)‐deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity‐dependent refinement of neuronal connections and normal synaptic plasticity. We found that β2m proteins were localised in hippocampal synapses in wild‐type mice. NMDA EPSCs in β2m‐deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25‐6981, an ɛ2 subunit‐selective antagonist, with those in ‘ɛ2‐dominant’ synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the β2m‐deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of ‘ɛ2‐dominant’ synapses. These observations indicate that the β2m‐deficient hippocampus lacks ‘ɛ2‐non‐dominant’ synapses and circuit asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.