diff --git a/src/node_dotenv.cc b/src/node_dotenv.cc
index d01538bd054ac6..59097806953ff8 100644
--- a/src/node_dotenv.cc
+++ b/src/node_dotenv.cc
@@ -142,6 +142,9 @@ void Dotenv::ParseContent(const std::string_view input) {
 
     // SAFETY: Content is guaranteed to have at least one character
     if (content.empty()) {
+      // In case the last line is a single key without value
+      // Example: KEY= (without a newline at the EOF)
+      store_.insert_or_assign(std::string(key), "");
       break;
     }
 
diff --git a/test/fixtures/dotenv/eof-without-value.env b/test/fixtures/dotenv/eof-without-value.env
new file mode 100644
index 00000000000000..1806141cfa01ae
--- /dev/null
+++ b/test/fixtures/dotenv/eof-without-value.env
@@ -0,0 +1,2 @@
+BASIC=value
+EMPTY=
\ No newline at end of file
diff --git a/test/parallel/test-dotenv-edge-cases.js b/test/parallel/test-dotenv-edge-cases.js
index 3a894270d9514e..f8cd262c8a8092 100644
--- a/test/parallel/test-dotenv-edge-cases.js
+++ b/test/parallel/test-dotenv-edge-cases.js
@@ -81,4 +81,21 @@ describe('.env supports edge cases', () => {
     assert.strictEqual(child.stderr, '');
     assert.strictEqual(child.code, 0);
   });
+
+  it('should handle empty value without a newline at the EOF', async () => {
+    // Ref: https://github.com/nodejs/node/issues/52466
+    const code = `
+      process.loadEnvFile('./eof-without-value.env');
+      require('assert').strictEqual(process.env.BASIC, 'value');
+      require('assert').strictEqual(process.env.EMPTY, '');
+    `.trim();
+    const child = await common.spawnPromisified(
+      process.execPath,
+      [ '--eval', code ],
+      { cwd: fixtures.path('dotenv') },
+    );
+    assert.strictEqual(child.stdout, '');
+    assert.strictEqual(child.stderr, '');
+    assert.strictEqual(child.code, 0);
+  });
 });