DNA polymerase λ (Pol λ) is the sole member of family X DNA polymerase in plants and plays a crucial role in nuclear DNA damage repair. Here, we report the transcriptional up-regulation of Arabidopsis (Arabidopsis thaliana) AtPolλ in response to abiotic and genotoxic stress, including salinity and the DNA cross-linking agent mitomycin C (MMC). The increased sensitivity of atpolλ knockout mutants toward high salinity and MMC treatments, with higher levels of accumulation of double strand breaks (DSBs) than wild-type plants and delayed repair of DSBs, has suggested the requirement of Pol λ in DSB repair in plants. AtPolλ overexpression moderately complemented the deficiency of DSB repair capacity in atpolλ mutants. Transcriptional up-regulation of major nonhomologous end joining (NHEJ) pathway genes KU80, X-RAY CROSS COMPLEMENTATION PROTEIN4 (XRCC4), and DNA Ligase4 (Lig4) along with AtPolλ in Arabidopsis seedlings, and the increased sensitivity of atpolλ-2/atxrcc4 and atpolλ-2/atlig4 double mutants toward high salinity and MMC treatments, indicated the involvement of NHEJ-mediated repair of salinity- and MMC-induced DSBs. The suppressed expression of NHEJ genes in atpolλ mutants suggested complex transcriptional regulation of NHEJ genes. Pol λ interacted directly with XRCC4 and Lig4 via its N-terminal breast cancer-associated C terminus (BRCT) domain in a yeast two-hybrid system, while increased sensitivity of BRCT-deficient Pol λ-expressing transgenic atpolλ-2 mutants toward genotoxins indicated the importance of the BRCT domain of AtPolλ in mediating the interactions for processing DSBs. Our findings provide evidence for the direct involvement of DNA Pol λ in the repair of DSBs in a plant genome.