Cardiac aging is sexually dimorphic, with women more likely than men to develop diastolic dysfunction (heart failure with preserved ejection fraction, HFpEF) for which no therapies exist. Loss of cardioprotective estrogen (E2) during menopause increases risk of HFpEF in women through unclear mechanisms. Expression of peptidylarginine deiminase 2 (PAD2) is positively regulated by E2, suggesting a potential novel mechanism linking PAD2, diastolic function, and E2 in the aging female heart. We hypothesized that PAD2 expression and protein citrullination decline with age in the female heart due to loss of E2, contributing to diastolic dysfunction. Global deletion of PAD2 exacerbated the HFpEF phenotype in aging females, with worse diastolic function than age matched controls. PAD2 expression and protein citrullination decreased with age in the female heart. Mass spectrometry detected citrullination of sarcomeric and metabolic proteins, with overall lower levels of citrullinated proteins in aged female hearts compared to young. To confirm direct regulation of PAD2 by E2, a cohort of young and aged mice underwent ovariectomy (OVX) with or without E2 replacement. Contrary to our hypothesis, no changes in PAD2 expression were observed in young females and PAD2 expression increased with OVX and OVX+E2 in aged females. Given the previous reports of hypoxia regulating PAD activity, we quantified expression of the transcriptional regulator hypoxia inducible factor-1α (HIF-1α). HIF-1α was upregulated by OVX and OVX+E2, suggesting a mechanism by which PAD2 is regulated by hypoxia. Together, we establish that protein citrullination and PAD2 in the female heart change with age, perhaps contributing to diastolic dysfunction. Elucidation of the mechanisms underlying PAD2 expression and function of citrullinated myocardial proteins remains to be determined and may benefit development of therapies for diastolic dysfunction in the aging female heart.