An ideal guided bone regeneration membrane (GBRM) is expected not only to perform barrier function, but also to enhance osteogenesis and resist bacteria infection. However, currently available membranes have limited bioactivities. To address this challenge, a Janus GBRM (JGM) is designed and fabricated by sequential fractional electrospinning here. The random gelatin fibers loaded with hydroxyapatite (HAP) are designed as the inner face to promote the osteoblasts' adhesion, proliferation, and osteogenic differentiation, meanwhile the aligned poly(caprolactone) (PCL) nanofibers loaded with poly(methacryloxyethyltrimethyl ammonium chloride-co-2-Aminoethyl 2-methylacrylate hydrochloride) (P(DMC-AMA)) are designed as the outer layer to resist epithelia invasion and bacterial infection. In vitro assays reveal that the inner face displays enhanced osteogenic effects, meanwhile the outer surface can regulate the epithelia to spread along the aligned direction and kill the contacted bacteria. Interestingly, the outer face can induce macrophages to polarize toward the M2 phenotype, thus manipulating a favorable osteoimmune environment. These results suggest that the JGM simultaneously meets the critical requirements of barrier, osteogenic, antibacterial, and osteoimmunomodulatory functions. Consequently, the JGM shows better in vivo bone tissue regeneration performance than the commercial Bio-Gide membrane. This work provides a novel platform to design multi-functional membranes/scaffolds, displaying great potential applications in tissue engineering.