diff --git a/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.conf b/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.conf
new file mode 100644
index 00000000000..9a83d7afb81
--- /dev/null
+++ b/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.conf
@@ -0,0 +1,4 @@
+# Copyright (c) 2024 Nordic Semiconductor ASA
+# SPDX-License-Identifier: Apache-2.0
+
+CONFIG_I2C_NRFX_TWIS_BUF_SIZE=256
diff --git a/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.overlay b/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.overlay
new file mode 100644
index 00000000000..928b52a66f5
--- /dev/null
+++ b/samples/drivers/i2c/rtio_loopback/boards/nrf54h20dk_nrf54h20_cpuapp.overlay
@@ -0,0 +1,71 @@
+/*
+ * Copyright (c) 2024 Nordic Semiconductor ASA
+ *
+ * SPDX-License-Identifier: Apache-2.0
+ */
+
+/*
+ * SDA = P2.8 and P2.9
+ * SCL = P1.2 and P1.3
+ */
+
+/ {
+	aliases {
+		i2c-controller = &i2c130;
+		i2c-controller-target = &i2c131;
+	};
+};
+
+&pinctrl {
+	i2c130_default: i2c130_default {
+		group1 {
+			psels = <NRF_PSEL(TWIM_SDA, 2, 8)>,
+				<NRF_PSEL(TWIM_SCL, 1, 2)>;
+			bias-pull-up;
+		};
+	};
+
+	i2c130_sleep: i2c130_sleep {
+		group1 {
+			psels = <NRF_PSEL(TWIM_SDA, 2, 8)>,
+				<NRF_PSEL(TWIM_SCL, 1, 2)>;
+			low-power-enable;
+		};
+	};
+
+	i2c131_default: i2c131_default {
+		group1 {
+			psels = <NRF_PSEL(TWIM_SDA, 2, 9)>,
+				<NRF_PSEL(TWIM_SCL, 1, 3)>;
+			bias-pull-up;
+		};
+	};
+
+	i2c131_sleep: i2c131_sleep {
+		group1 {
+			psels = <NRF_PSEL(TWIM_SDA, 2, 9)>,
+				<NRF_PSEL(TWIM_SCL, 1, 3)>;
+			low-power-enable;
+		};
+	};
+};
+
+&i2c130 {
+	clock-frequency = <I2C_BITRATE_STANDARD>;
+	pinctrl-0 = <&i2c130_default>;
+	pinctrl-1 = <&i2c130_sleep>;
+	pinctrl-names = "default", "sleep";
+	zephyr,concat-buf-size = <256>;
+	memory-regions = <&cpuapp_dma_region>;
+	status = "okay";
+};
+
+&i2c131 {
+	compatible = "nordic,nrf-twis";
+	clock-frequency = <I2C_BITRATE_STANDARD>;
+	pinctrl-0 = <&i2c131_default>;
+	pinctrl-1 = <&i2c131_sleep>;
+	pinctrl-names = "default", "sleep";
+	memory-regions = <&cpuapp_dma_region>;
+	status = "okay";
+};
diff --git a/samples/drivers/i2c/rtio_loopback/sample.yaml b/samples/drivers/i2c/rtio_loopback/sample.yaml
index 4234c090c67..68a752df947 100644
--- a/samples/drivers/i2c/rtio_loopback/sample.yaml
+++ b/samples/drivers/i2c/rtio_loopback/sample.yaml
@@ -12,3 +12,4 @@ tests:
       - b_u585i_iot02a
       - nrf5340dk/nrf5340/cpuapp
       - nrf54l15dk/nrf54l15/cpuapp
+      - nrf54h20dk/nrf54h20/cpuapp