In living organisms, uroporphyrinogen I occurs as a side branch of the main porphyrin synthesis pathway.

In the normal pathway, the linear tetrapyrrole precursor preuroporphyrinogen (a substituted hydroxymethylbilane) is converted by the enzyme uroporphyrinogen-III cosynthase into the cyclic uroporphyrinogen III; which is then converted to coproporphyrinogen III on the way to porphyrins like heme.

Uroporphyrinogen I is instead produced spontaneously from preuroporphyrinogen when the enzyme is not present.

The non-enzymatic conversion to uroporphyrinogen I results in the sequence AP-AP-AP-AP, whereas the enzymatic conversion into uroporphyrinogen III leads to reversal of one AP-group and hence an AP-AP-AP-PA arrangement.

If synthesized, uroporphyrinogen I is then converted into coproporphyrinogen I by the same enzyme (uroporphyrinogen decarboxylase) that acts on the III form; but that product, which is cytotoxic, then accumulates causing the pathology congenital erythropoietic porphyria.