In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria‐associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER‐anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid‐like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51–VAPB interaction is mediated by the FFAT‐like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM‐tethering function. SYNOPSIS The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity. The crystal structure of the TPR domain of PTPIP51 at 1.45 Å resolution revealed the presence of a lipid‐like serpentine electron density. PTPIP51 has phospholipid (especially phosphatidic acid) binding and transfer functions in vitro. Mitochondrial cardiolipin levels are affected by PTPIP51. The crystal structure and biochemical analyses of PTPIP51, a mitochondrial protein localized at the mitochondria‐associated ER membrane (MAM), revealed its phospholipid binding and transfer activity.