Oxytocin (OXT) is a neuropeptide known for modulating anxiety and fear memory. We have reported that microglial cytokine regulates contextual fear memory and that microglial OXT positively correlates with cytokine secretion. However, the relationship between contextual fear memory and microglial OXT expression remains unclear. We evaluated whether experimental handling minimizes anxiety-like behaviors through microglial OXT expression and its effects on contextual fear response in a sex-dependent manner. Male and female mice were cup-handled for seven days by male or female experimenters (four groups: male mice with or without handling and female mice with or without handling). Post-handling anxiety-like behavior was assessed using elevated plus maze (EPM) and light-dark box (LDB) tests. Microglial Oxt transcription was evaluated using real-time PCR following handling and footshock. Our results showed that handling by female experimenters induced anxiolytic behaviors in the EPM and LDB and microglial Oxt transcripts in male mice but did not show a direct causal relationship. After handling by male experimenters, male mice exhibited stronger conditional freezing responses than female mice. In contrast, female mice exhibited significantly weaker freezing, independent of Oxt transcription in the microglia and the paraventricular hypothalamic nucleus. These findings suggest that handling influences anxiety and microglial Oxt expression, while conditional freezing reflects a sex-dependent effect by experimenter sex.