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In 1999, development on a successor to the W163 began under the codename "W164" and spanned a period of 6 years.

First design drafts have appeared in 2000 with the first production models in scale 1:4, and in 2001, three full-sized versions were prepared. By late 2001, the production design styled by Steve Mattin under Peter Pfeiffer was chosen and approved by the executive board in 2002.

Prototype testing was conducted throughout 2003 and 2004, concluding in early 2005. The redesigned M-class (chassis name W164) was introduced in April 2005 as a 2006 model after a showing at the North American International Auto Show in January. It was almost entirely new, with a more sporting, aerodynamic look—the coefficient of drag went down to 0.34 compared with the previous generation. Mercedes-Benz made the new M-Class 71 mm larger, 150 mm longer and 5 mm lower than the first model.

Mercedes-Benz extensively publicized the $600 million spent to update its factory and add manufacturing space for the new R-Class. According to early automotive press reports, the 2006 M-Class vehicles demonstrated vast improvements in quality.

The W164 platform used for the new M-Class is shared with the new GL-Class and is a unibody type rather than the former (W163) body-on-frame used by the M-Class vehicles produced from 1998–2005. The X164 GL-Class, a longer seven-seater version of the W164 platform, is also available.

New features in the 2006 M-Class include the 7G-Tronic seven-speed automatic transmission, optional Active Curve-Illuminating Bi-Xenon headlights which "steer" in the path of the vehicle, and an adjustable-height air suspension. The manual transmission has been dropped. Permanent 4Matic four-wheel drive remained, with one-speed transfer case (no low range), center limited-slip differential and four-wheel electronic traction system (4-ETS). In most countries except the U.S., an Off-Road Pro package with center and rear differential locks, 4-ETS, two-speed transfer case with reduction gearing, and adjustable ground clearance was available as an option increasing the original fording depth from 500 mm to 600 mm.

The AMG version of the W164, the ML 63 AMG, was introduced at the 2006 North American International Auto Show as a limited edition 2007 model. It features a 6.2 L M156 V8 engine producing 503 HP and 465 lb⋅ft of torque that is handcrafted in Germany. The engine is added to an AMG Speedshift 7G-Tronic seven-speed automatic transmission. The 2006 ML 63 AMG can accelerate from 0–60 mph (96 km/h) in just 4.8 seconds, or from 0–62 mph (100 km/h) in just 5.0 seconds. These impressive features made the ML 63 AMG the most powerful naturally aspirated V8 SUV in the world.