Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by lower lip pits and orofacial clefts (OFCs). With a prevalence of approximately 1 in 35,000 live births, it is the most common form of syndromic clefting and may account for ~2% of all OFCs. The majority of VWS is attributed to genetic variants in IRF6 (~70%) or GRHL3 (~5%), leaving up to 25% of individuals with VWS without a molecular diagnosis. Both IRF6 and GRHL3 function in a transcriptional regulatory network governing differentiation of periderm, a single layer of epithelial cells that prevents pathological adhesions during palatogenesis. Disruption of this layer results in a spectrum of phenotypes ranging from lip pits and OFCs to severe pterygia and other congenital anomalies that can be incompatible with life. Understanding the mechanisms of peridermopathies is vital in improving health outcomes for affected individuals. We reasoned that genes encoding additional members of the periderm gene regulatory network, including kinases acting upstream of IRF6 (i.e., atypical protein kinase C family members, RIPK4, and CHUK), are candidates to harbor variants resulting in VWS. Consistent with this prediction, we identified 6 de novo variants (DNs) and 11 rare variants in PRKCI, an atypical protein kinase C, in 17 individuals with clinical features consistent with syndromic OFCs and peridermopathies. Of the identified DNs, 4 were identical p.(Asn383Ser) variants in unrelated individuals with syndromic OFCs, indicating a likely hotspot mutation. We also performed functional validation of 12 variants using the enveloping layer in zebrafish embryos, a structure analogous to the periderm. Three patient-specific alleles (p.Arg130His, p.(Asn383Ser), and p.Leu385Phe) were confirmed to be loss-of-function variants. In summary, we identified PRKCI as a novel causal gene for VWS and syndromic OFC with other features of peridermopathies.