Cell proliferation can be dependent on the non-essential amino acid serine, and dietary restriction of serine inhibits tumor growth, but the underlying mechanisms remain incompletely understood. Using a metabolomics approach, we found that serine deprivation most predominantly impacts cellular acylcarnitine levels, a signature of altered mitochondrial function. Fuel utilization from fatty acid, glucose, and glutamine is affected by serine deprivation, as are mitochondrial morphological dynamics leading to increased fragmentation. Interestingly, these changes can occur independently of nucleotide and redox metabolism, two known major functions of serine. A lipidomics analysis revealed an overall decrease in ceramide levels. Importantly, supplementation of the lipid component of bovine serum or C16:0-ceramide could partially restore defects in cell proliferation and mitochondrial fragmentation induced by serine deprivation. Together, these data define a role for serine in supporting mitochondrial function and cell proliferation through ceramide metabolism.