This review is focused on recent data on the role of proline (Pro) in collagen biosynthesis and cellular metabolism. It seems obvious that one of the main substrates for collagen biosynthesis Pro is required to form collagen molecule. The question raised in this review is whether the Pro for collagen biosynthesis is synthesized “de novo”, comes directly from degraded proteins or it is converted from other amino acids. Recent data provided evidence that extracellular Pro (added to culture medium) had significant, but relatively little impact on collagen biosynthesis in fibroblasts (the main collagen synthesized cells) cultured in the presence of glutamine (Gln). However, extracellular Pro drastically increased collagen biosynthesis in the cells cultured in Gln-free medium. It suggests that Pro availability determines the rate of collagen biosynthesis and demand for Pro in fibroblasts is predominantly met by conversion from Gln. The potential mechanism of this process as well as possible implication of this knowledge in pharmacotherapy of connective tissue diseases is discussed in this review.