Reproducing the native collagen structure and glycosaminoglycan (GAG) distribution in tissue-engineered cartilage constructs is still a challenge. Articular cartilage has a specific nutrient supply and mechanical environment due to its location and function in the body. Efforts to simulate this native environment have been reported through the use of bioreactor systems. However, few of these devices take into account the existence of gradients over cartilage as a consequence of the nutrient supply by diffusion. We hypothesized that culturing chondrocytes in an environment, in which gradients of nutrients can be mimicked, would induce zonal differentiation. Indeed, we show that glucose gradients facilitating a concentration distribution as low as physiological glucose levels enhanced a zonal chondrogenic capacity similar to the one found in native cartilage. Furthermore, we found that the glucose consumption rates of cultured chondrocytes were higher under physiological glucose concentrations and that GAG production rates were highest in 5 mM glucose. From these findings, we concluded that this condition is better suited for matrix deposition compared to 20 mM glucose standard used in a chondrocyte culture system. Reconsidering the culture conditions in cartilage tissue engineering strategies can lead to cartilaginous constructs that have better mechanical and structural properties, thus holding the potential of further enhancing integration with the host tissue.